Intermyofibrillar Research Articles

Tom40 import and TOM complex assembly kinetics in subsarcolemmal and intermyofibrillar mitochondria

The insertion of nuclear-derived proteins into mitochondria is facilitated by the translocases of the outer membrane (TOM) complex. Tom40 is the main component of this complex. Its import and assembly has not been characterized in skeletal muscle. Thus, our purpose was to compare the import and assembly of Tom40 in muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. Tom40 protein levels were not different between SS and IMF mitochondria. However, Tom40 import into the outer membrane was higher (p<0.05) in SS mitochondria at all time points (5, 10, 20 min) when compared to IMF mitochondria. Tom40 assembly was monitored using blue native electrophoresis. Tom40 first assembles into a ~250kDa complex (Intermediate I), followed by a ~120 kDa complex (Intermediate II), before its final insertion into the ~380 kDa TOM complex. In IMF mitochondria, 70–80% of Tom40 was incorporated into Intermediate I by 5 min, whereas only 40–50% was found in Intermediate I in SS mitochondria. However, the sequential incorporation of Tom40 into Intermediate II was less rapid in IMF mitochondria, and only 45% of Tom40 was incorporated into the final TOM complex by 60 min. In contrast, SS mitochondria incorporated 65–70% of Tom40 by 60 min. Thus, despite similar levels of steady state Tom40 protein content, the import and assembly of Tom40 into the TOM complex differs among muscle mitochondrial subtypes.

Myocardial subsarcolemmal and intermyofibrillar mitochondria respond differently to apoptotic stimuli

Reactive oxygen species (ROS) can trigger apoptosis by increasing permeability of the mitochondrial permeability transition pore (mtPTP) leading to release of the proapoptotic factors cytochrome c and apoptosis-inducing factor (AIF). Two subpopulations of mitochondria exist in cardiac muscle, but it is unknown if these mitochondrial subpopulations differ in their susceptibility to apoptotic stimuli. Therefore, we investigated the susceptibility of cardiac subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subfractions to apoptosis-induced oxidative stress. Basal protein levels of key mtPTP components (voltage-dependent anion channel 1, adenine nucleotide translocase, and cyclophilin D) were higher in SS compared to IMF mitochondria. In response to apoptotic stimuli, IMF mitochondria exhibit a higher rate of mtPTP opening (Vmax), but time required to reach Vmax was shorter for SS mitochondria. Exposure to ROS induced an increase in cytochrome c and AIF release from IMF mitochondria, but did not alter release in SS mitochondria. We conclude that cardiac muscle SS and IMF mitochondria differentially respond to apoptotic stimuli. Supported by National Institutes of Health grant R01HL067855 awarded to S.K. Powers.

Effect of denervation on mitochondrially mediated apoptosis in skeletal muscle

Chronic muscle disuse induced by denervation reduces mitochondrial content and produces muscle atrophy. To investigate the molecular mechanisms responsible for these adaptations, we assessed 1) mitochondrial biogenesis- and apoptosis-related proteins and 2) apoptotic susceptibility and cell death following denervation. Rats were subjected to 5, 7, 14, 21, or 42 days of unilateral denervation of the sciatic or peroneal nerve. Muscle mass and mitochondrial content were reduced by 40-65% after 21 and 42 days of denervation. Denervation-induced decrements in mitochondrial content occurred along with 60% and 70% reductions in transcription factor A (Tfam) and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha, respectively. After 42 days of denervation, Bax was elevated by 115% and Bcl-2 was decreased by 89%, producing a 16-fold increase in the Bax-to-Bcl-2 ratio. Mitochondrial reactive oxygen species production was markedly elevated by 5- to 7.5-fold in subsarcolemmal mitochondria after 7, 14, and 21 days of denervation, whereas reactive oxygen species production in intermyofibrillar (IMF) mitochondria was reduced by 40-50%. Subsarcolemmal and IMF mitochondrial levels of MnSOD were also reduced by 40-50% after 14-21 days of denervation. The maximal rate of IMF mitochondrial pore opening (V(max)) was elevated by 25-35%, and time to V(max) was reduced by 20-25% after 14 and 21 days, indicating increased apoptotic susceptibility. Myonuclear decay, assessed by DNA fragmentation, was elevated at 7-21 days of denervation. Our data indicate that PGC-1alpha and Tfam are important factors that likely contribute to the reduced mitochondrial content after chronic disuse. In addition, our results illustrate that, despite the reduced mitochondrial content, denervated muscle has greater mitochondrial apoptotic susceptibility, which coincided with elevated apoptosis, and these processes may contribute to denervation-induced muscle atrophy.

Effect of chronic contractile activity on SS and IMF mitochondrial apoptotic susceptibility in skeletal muscle

Chronic contractile activity of skeletal muscle induces an increase in mitochondria located in proximity to the sarcolemma [subsarcolemmal (SS)] and in mitochondria interspersed between the myofibrils [intermyofibrillar (IMF)]. These are energetically favorable metabolic adaptations, but because mitochondria are also involved in apoptosis, we investigated the effect of chronic contractile activity on mitochondrially mediated apoptotic signaling in muscle. We hypothesized that chronic contractile activity would provide protection against mitochondrially mediated apoptosis despite an elevation in the expression of proapoptotic proteins. To induce mitochondrial biogenesis, we chronically stimulated (10 Hz; 3 h/day) rat muscle for 7 days. Chronic contractile activity did not alter the Bax/Bcl-2 ratio, an index of apoptotic susceptibility, and did not affect manganese superoxide dismutase levels. However, contractile activity increased antiapoptotic 70-kDa heat shock protein and apoptosis repressor with a caspase recruitment domain by 1.3- and 1.4-fold (P<0.05), respectively. Contractile activity elevated SS mitochondrial reactive oxygen species (ROS) production 1.4- and 1.9-fold (P<0.05) during states IV and III respiration, respectively, whereas IMF mitochondrial state IV ROS production was suppressed by 28% (P<0.05) and was unaffected during state III respiration. Following stimulation, exogenous ROS treatment produced less cytochrome c release (25-40%) from SS and IMF mitochondria, and also reduced apoptosis-inducing factor release (approximately 30%) from IMF mitochondria, despite higher inherent cytochrome c and apoptosis-inducing factor expression. Chronic contractile activity did not alter mitochondrial permeability transition pore (mtPTP) components in either subfraction. However, SS mitochondria exhibited a significant increase in the time to Vmax of mtPTP opening. Thus, chronic contractile activity induces predominantly antiapoptotic adaptations in both mitochondrial subfractions. Our data suggest the possibility that chronic contractile activity can exert a protective effect on mitochondrially mediated apoptosis in muscle.

Functional Characteristics of Subsarcolemmal and Intermyofibrillar Mitochondria Isolated from H-FABP Deficient Mice

Subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria are two morphologically distinct subfractions located in different regions of the myofiber, possessing disparate biochemical properties which may contribute to their adaptive potentials. Although mice lacking the skeletal muscle cytosolic fatty acid (FA) transporter heart-type fatty acid-binding protein (H-FABP) exhibit abnormal FA metabolism, it is unknown how SS and IMF mitochondria adapt functionally to this deficit. PURPOSE: To investigate the composition and function of SS and IMF mitochondria isolated from WT compared to H-FABP KO animals. METHODS: SS and IMF mitochondria were isolated from muscle of WT and H-FABP KO animals and cardiolipin content was assessed using flow cytometry. State 3 (active) and state 4 (passive) mitochondrial oxygen consumption (VO2) was measured. Fluorometric and flow cytometric techniques were used to measure mitochondrial reactive oxygen species (ROS) generation and membrane potential. Mitochondrial permeability transition pore (mtPTP) opening kinetics, including the maximal rate of pore opening (Vmax) and the time required to achieve Vmax, were also assessed. RESULTS: Cardiolipin content was similar between SS and IMF mitochondria isolated from WT and H-FABP KO mice. IMF mitochondrial VO2 was 1.5-and 2-fold greater compared to SS during state 4 and state 3 respiration, respectively. IMF mitochondrial ROS production, along with membrane potential, were 35-70% lower compared to the levels in SS mitochondria in both WT and H-FABP KO mice. ROS generation during SS state 3 respiration was 30% higher in H-FABP KO compared to WT animals. mtPTP Vmax was 2.7-fold higher in IMF mitochondria compared to the SS subfraction isolated from WT and H-FABP KO mice. Time to Vmax in IMF mitochondria harvested from H-FABP KO mice was 40% greater compared to WT littermates. In addition, within the H-FABP deficient genotype, time to Vmax was 66% higher for the IMF subfraction compared to SS mitochondria. CONCLUSION: These findings reveal unique adaptive strategies between SS and IMF mitochondria due to an alteration in FA handling. The data demonstrate an association between high rates of mitochondrial VO2, low membrane potential, and reduced levels of ROS production in IMF mitochondria, while the inverse appears to hold for the SS subfraction. Further, SS mitochondria upregulate ROS production as a potential signal to increase the transcription of other genes, possibly involved in FA metabolism. The IMF subfraction demonstrates a reduced susceptibility to mitochondrially-mediated apoptosis, a protective adaptation to possible lipotoxic conditions within the myocyte.

Altered Skeletal Muscle Subsarcolemmal Mitochondrial Compartment During Catch-Up Fat After Caloric Restriction

An accelerated rate of fat recovery (catch-up fat) and insulin resistance are characteristic features of weight recovery after caloric restriction, with implications for the pathophysiology of catch-up growth and weight fluctuations. Using a previously described rat model of weight recovery in which catch-up fat and skeletal muscle insulin resistance have been linked to suppressed thermogenesis per se, we investigated alterations in mitochondrial energetics and oxidative stress in subsarcolemmal (SS) and intermyofibrillar (IMF) skeletal muscle mitochondria. After 2 weeks of semistarvation followed by 1 week of refeeding, the refed rats show persistent and selective reductions in SS mitochondrial mass (assessed from citrate synthase activity in tissue homogenate and isolated mitochondria) and oxidative capacity. Furthermore, the refed rats show, in both SS and IMF muscle mitochondria, a lower aconitase activity (whose inactivation is an index of increased reactive oxygen species [ROS]), associated with higher superoxide dismutase activity and increased proton leak. Taken together, these studies suggest that diminished skeletal muscle mitochondrial mass and function, specifically in the SS mitochondrial compartment, contribute to the high metabolic efficiency for catch-up fat after caloric restriction and underscore a potential link between diminished skeletal muscle SS mitochondrial energetics, increased ROS concentration, and insulin resistance during catch-up fat.

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.

Adaptation of skeletal muscle pyruvate dehydrogenase kinase in response to starvation in mitochondrial subpopulations

Pyruvate dehydrogenase (PDH) catalyses decarboxylation of pyruvate, to form acetyl-CoA. PDH activity is down-regulated by intrinsic PDH kinases (predominantly PDK2 and PDK4 isoforms), but the understanding of the PDK isoform distribution and adaptation to nutritional stresses has been restricted to mixed mitochondrial populations, and not delineated between subsarcolemmal (SS) and intermyofibrillar (IMF) sub-populations. SS and IMF exhibit distinct morphological and biochemical properties; however the functional differences are not well understood. This study investigated the effect of fed (FED) versus 48 h food restriction (FR) on adaptive changes in rat red gastrocnemius muscle PDK2 and 4 isoform content in SS and IMF mitochondria. PDK4 content was 2–4 fold higher in SS mitochondria compared to IMF (p=0.003), and increased with FR ~10- fold in both sub-populations (p<0.001). PDK2 was ~3–4 fold higher in SS mitochondria compared to IMF (p=0.017), but PDK2 was unaltered with FR. These results demonstrate that there is a markedly higher content of both PDK isoforms in SS compared to IMF mitochondria. Although PDK2 does not increase in either sub-population in response to FR, PDK4 increases to a similar extent in both SS and IMF after 48 h food restriction Supported by NSERC (SJP, BDR & PJL) and CIHR (LLM).

Effect of denervation on mitochondrial function and the expression of apoptotic related proteins

Chronic skeletal muscle disuse results in a loss of muscle mass which is partly attributable to apoptosis. This study was designed to evaluate the relationship between changes in mitochondrial function and apoptosis in chronic denervation (7, 14, 21 days) of rat tibialis anterior (TA) muscle. Denervation decreased state 3 (ADP-stimulated) respiration in subsarcolemmal (SS) mitochondria by 37 – 45 % between 7 and 21 days, whereas no changes were evident in intermyofibrillar (IMF) mitochondria. Conversely, state 4 respiration increased by 68% in IMF mitochondria with 14 days of denervation, while it remained unchanged in the SS mitochondria. This was accompanied by a 2.3-fold increase in UCP3 expression at 14 days of denervation. The expression of the anti-oxidant enzyme MnSOD declined 60–70% in SS and IMF mitochondria, whereas the production of reactive oxygen species (ROS) was elevated 5–10-fold in the SS mitochondrial subfraction. In addition, there was a 76% increase in the expression of the pro-apoptotic protein p53 at 14 days of denervation. This was matched by an increase in DNA fragmentation, assessed using TUNEL staining. An elevation in apoptotic nuclei was evident as early as 7 days, which remained similarly elevated at 14 and 21 days. Thus chronic muscle denervation: 1) induces impairments in mitochondrial function, leading to increased ROS production; and 2) increases the expression of pro-apoptotic proteins while reducing the protective effects of anti-oxidant enzymes. These data suggest that mitochondrially mediated apoptosis contributes to the muscle atrophy which is observed during muscle disuse.

Regulation of Fatty Acid Transport Across the Mitochondrial Membranes in Human and Rodent Skeletal Muscle

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Differential susceptibility of subsarcolemmal and intermyofibrillar mitochondria to apoptotic stimuli

Apoptosis can be evoked by reactive oxygen species (ROS)-induced mitochondrial release of the proapoptotic factors cytochrome c and apoptosis-inducing factor (AIF). Because skeletal muscle is composed of two mitochondrial subfractions that reside in distinct subcellular regions, we investigated the apoptotic susceptibility of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS and IMF mitochondria exhibited a dose-dependent release of protein in response to H2O2 (0, 25, 50, and 100 microM). However, IMF mitochondria were more sensitive to H2O2 and released a 2.5-fold and 10-fold greater amount of cytochrome c and AIF, respectively, compared with SS mitochondria. This finding coincided with a 44% (P < 0.05) greater rate of opening (maximum rate of absorbance decrease, V(max)) of the protein release channel, the mitochondrial permeability transition pore (mtPTP), in IMF mitochondria. IMF mitochondria also exhibited a 47% (P < 0.05) and 60% (0.05 < P < 0.1) greater expression of the key mtPTP component voltage-dependent anion channel and cyclophilin D, respectively, along with a threefold greater cytochrome c content, but similar levels of AIF compared with SS mitochondria. Despite a lower susceptibility to H2O2-induced release, SS mitochondria possessed a 10-fold greater Bax-to-Bcl-2 ratio (P < 0.05), a 2.7-fold greater rate of ROS production, and an approximately twofold greater membrane potential compared with IMF mitochondria. The expression of the antioxidant enzyme Mn2+-superoxide dismutase was similar between subfractions. Thus the divergent protein composition and function of the mtPTP between SS and IMF mitochondria contributes to a differential release of cytochrome c and AIF in response to ROS. Given the relatively high proportion of IMF mitochondria within a muscle fiber, this subfraction is likely most important in inducing apoptosis when presented with apoptotic stimuli, ultimately leading to myonuclear decay and muscle fiber atrophy.

Regional Distribution Of SDH In Single Muscle Fibres After Training In Endurance Cyclists

Previous research has shown that two populations of mitochondria with different metabolic properties exist in human skeletal muscle. Recent research has shown that the regional distribution of succinate dehydrogenase (SDH) in single muscle fibres is altered with moderate intensity endurance training. Based on this information, we tested the hypothesis that the regional distribution of mitochondria would be altered following sustained high intensity endurance training. PURPOSE The purpose of this study was to examine the changes in the subsarcolemmal (SSM) and intermyofibrillar (IMF) mitochondria as represented by changes in SDH activity. METHODS Eight (2 female, 6 male) trained cyclists (VO2max = 60.7 mL.kg−1·min−1) performed eight weeks of high intensity endurance training for 60 minutes × 5d.wk−1 at 80–85% VO2max. Pre- and post-training muscle biopsy samples were taken from the vastus lateralis, and then immediately frozen in isopentane until analyzed histochemically for SDH activity. Frozen tissue sections (6μm) were incubated and then analyzed using a computer-enhanced image analysis system which digitized selected fibres (18–75) to represent SDH activity as an optical density. Concentric rings (pixel width 0.75μm) were partitioned from the outside layer (SSM, <5.25μm) to the inside layer (IMF, > 6.00μm). Based on myofibrillar ATPase staining, Type I and Type II fibres were identified. RESULTS The results showed that significant changes (ANOVA, p < 0.05) occurred for all analyses performed, both pre- vs. post-training for fibre types, and between SSM and IMF mitochondria. SDH activity increased significantly in Type II (71.7%) vs. Type I (28.3%) over training. The greatest increases in the mitochondrial activity occurred in the SSM (54.6%) vs. IMF (45.4%). However, the greatest increase in SDH activity occurrred in the IMF mitochondria of Type II fibres (39.7%). CONCLUSIONS These data demonstrated that high intensity endurance training can elicit selective adaptations in the metabolic profile of different fibre types in single muscle fibres, and that regional differences in SDH activity can occur.

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, Ca 2+-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.

CARNITINE PALMITOYLTRANSFERASE I ACTIVITY IS UNAFFECTED BY CALCIUM AND ENERGY CHARGE METABOLITES IN HUMAN SKELETAL MUSCLE MITOCHONDRIA

Carnitine palmitoyltransferase I (CPTI) activity regulates the transport of long chain fatty acids into the mitochondria and is inhibited by malonyl-CoA (M-CoA) in vivo. Little is known regarding CPTI regulation in human skeletal muscle during exercise. PURPOSE To investigate the effects of physiological concentrations of calcium and energy charge metabolites on CPTI activity in intermyofibrillar (IMF) and subsarcolemmal (SS) populations of mitochondria. METHODS Nine recreationally active males volunteered for this study. IMF and SS mitochondria were extracted from the vastus lateralis muscle by homogenization and differential centrifugation. Muscle concentrations of M-CoA, calcium, and free AMP, ADP and inorganic phosphate (Pi) representative of 65% VO2max were added to IMF and SS mitochondrial preparations. CPTI activity was measured. RESULTS Maximal CPTI activity was significantly higher in IMF vs. SS mitochondria (325 ± vs. 233 ± 25, P = 0.018 nmol/min/g wm). Citrate synthase was similar in both mitochondrial fractions (2.0 ± 0.3 vs. 2.1 ± 0.3 nmol/min/g in IMF vs. SS). Addition of a physiological concentration of M-CoA (0.7 μ M) resulted in a similar inhibition of CPTI activity in IMF vs. SS mitochondria (29.9 ± 5.8% vs. 34.4 ± 5.3%). The addition of calcium, and free AMP, ADP and Pi in the presence of 0.7 μ M M-CoA had no significant effect on CPTI activity when compared to 0.7 μ M M-CoA alone in both fractions. However there was a trend (P = 0.098) for ADP to release the M-CoA inhibition in the IMF fraction. CONCLUSION Maximal CPTI activity is higher in IMF than SS mitochondria, however M-CoA sensitivity between the fractions is similar. The results suggest that calcium and energy charge metabolites do not increase CPTI activity during exercise in human skeletal muscle. Supported by NSERC & CIHR, Canada.

Expression of uncoupling protein-3 in subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its modulation by fasting.

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.

EFFECT OF CONTRACTILE ACTIVITY ON SUBSARCOLEMMAL AND INTERMYOFIBRILLAR MITOCHONDRIAL RESPIRATION AND NF-KB DNA BINDING

Skeletal muscle contains two morphologically distinct subfractions of mitochondria which are found in separate locations in the cell. Subsarcolemmal (SS) mitochondria are located beneath the surface of the sarcolemma and intermyofibrillar (IMF) mitochondria are found intermingled within the myofibrils. SS and IMF subfractions possess different enzyme activities and respiratory rates, and they adapt differently to contractile activity. The purpose of this study was to determine if there is a differential adaptive response of IMF and SS mitochondrial respiration to chronic stimulation (1,2,3,5,7 days, 10Hz, 3hrs/d, n = 2–7/day) of rat tibialis anterior muscles. Since oxygen consumption is associated with the production of reactive oxygen species (ROS), and these are known activators of the transcription factor NF-kB, we also measured NF-kB-DNA binding using electromobility shift assays (n = 4/day). Following 1 day of stimulation, SS mitochondrial state 3 and 4 respiration rates were transiently decreased by 40–50% in stimulated, compared to control muscle. This was followed by a return to control levels of state 4 respiration, and a dramatic 63% increase in state 3 respiration above control levels between 3–5d of stimulation. SS mitochondrial state 3 respiration returned to control levels by 7d. In contrast, IMF mitochondrial state 3 and 4 respiration rates were not changed between 1–5d, but increased in parallel by 35–40% after 7d of stimulation. Electromobility shift assays revealed elevated levels of NF-kB binding above that in control muscle extracts by 40% after 1d, 130% after 5d, and 80% after 7d (n = 4) of stimulation. These data indicate chronic contractile activity induces early increases in NF-kB binding which precede changes in muscle respiratory capacity. In addition, contractile activity induces differential adaptive responses with respect to IMF and SS mitochondrial respiration rates.

Dietary coconut oil affects more lipoprotein lipase activity than the mitochondria oxidative capacities in muscles of preruminant calves

The presence of coconut oil in a milk replacer stimulates the growth rate of calves, suggesting a better oxidation of fatty acid in muscles. Because dietary fatty acid composition influences carnitine palmitoyltransferase I (CPT I) activity in rat muscles, this study was designed to examine the effects of a milk replacer containing either tallow (TA) or coconut oil (CO) on fatty acid utilization and oxidation and on the characteristics of intermyofibrillar (IM) and subsarcolemmal (SS) mitochondria in the heart and skeletal muscles of preruminant calves. Feeding CO did not affect palmitate oxidation rate by whole homogenates, but induced higher palmitate oxidation by IM mitochondria (+37%, P < 0.05). CPT I activity did not significantly differ between the two groups of calves. Heart and longissimus thoracis muscle of calves fed CO had higher lipoprotein lipase activity (+27% and 58%, respectively; P < 0.05) but showed no differences in fatty acid binding protein content or activity of oxidative enzymes. Whatever the muscle and the diet, IM mitochondria had higher respiration rates and enzyme activities than those of SS mitochondria ( P < 0.05). Furthermore, CPT I activity of the heart was 28-fold less sensitive to malonyl-coenzyme A inhibition in IM mitochondria than in SS mitochondria. In conclusion, dietary CO marginally affected the activity of the two mitochondrial populations and the oxidative activity of muscles in the preruminant calf. In addition, this study showed that differences between IM and SS mitochondria in the heart and muscles were higher in calves than in other species studied so far.

Differential responses to endurance training in subsarcolemmal and intermyofibrillar mitochondria.

To examine the effect of endurance training (6 wk of treadmill running) on regional mitochondrial adaptations within skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria were isolated from trained and control rat hindlimb muscles. Mitochondrial oxygen consumption (VO2) was measured polarographically by using the following substrates: 1 mM pyruvate + 1 mM malate (P+M), 10 mM 2-oxoglutarate, 45 microM palmitoyl-DL-carnitine + 1 mM malate, and 10 mM glutamate. Spectrophotometric assays of cytochrome-c reductase and NAD-specific isocitrate dehydrogenase (IDH) activity were also performed. Maximal (state III) and resting (state IV) VO2 were lower in SS than in IMF mitochondria in both trained and control groups. In SS mitochondria, training elicited significant 36 and 20% increases in state III VO2 with P+M and glutamate, respectively. In IMF mitochondria, training resulted in a smaller (20%), yet significant, increase in state III VO2 with P+M as a substrate, whereas state III VO2 increased 33 and 27% with 2-oxoglutarate and palmitoyl-DL-carnitine + malate, respectively. Within groups, cytochrome-c reductase and IDH activities were lower in SS when compared with IMF mitochondria. Training increased succinate-cytochrome-c reductase in both SS (30%) and IMF mitochondria (28%). IDH activity increased 32% in the trained IMF but remained unchanged in SS mitochondria. We conclude that endurance training promotes substantial changes in protein stoichiometry and composition of both SS and IMF mitochondria.

Higher mitochondrial fatty acid oxidation following intermittent versus continuous endurance exercise training

It has been well documented that skeletal muscle fatty acid oxidation can be elevated by continuous endurance exercise training. However, it remains questionable whether similar adaptations can be induced with intermittent interval exercise training. This study was undertaken to directly compare the rates of fatty acid oxidation in isolated subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria following these different exercise training regimes. Mitochondria were isolated from the gastrocnemius-plantaris muscles of male Sprague-Dawley rats following exercise training 6 days per week for 12 weeks. Exercise training consisted of either continuous, submaximal, endurance treadmill running (n = 10) or intermittent, high intensity, interval running (n = 10). Both modes of training enhanced the oxidation of palmityl-carnitine-malate in both mitochondrial populations (p &lt; 0.05). However, the increase associated with the intermittent, high intensity exercise training was significantly greater than that achieved with the continuous exercise training (p &lt; 0.05). Also, the increases associated with the IMF mitochondria were greater than the SS mitochondria (p &lt; 0.05). These data suggest that high intensity, intermittent interval exercise training is more effective for stimulation of fatty acid oxidation than continuous submaximal exercise training and that this adaptation occurs preferentially within IMF mitochondria.Key words: muscle, subsarcolemmal mitochondria, intermyofibrillar mitochondria.

Contractile activity-induced adaptations in the mitochondrial protein import system.

We previously demonstrated that subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subfractions import proteins at different rates. This study was undertaken to investigate 1) whether protein import is altered by chronic contractile activity, which induces mitochondrial biogenesis, and 2) whether these two subfractions adapt similarly. Using electrical stimulation (10 Hz, 3 h/day for 7 and 14 days) to induce contractile activity, we observed that malate dehydrogenase import into the matrix of the SS and IMF mitochondia isolated from stimulated muscle was significantly increased by 1.4-to 1.7-fold, although the pattern of increase differed for each subfraction. This acceleration of import may be mitochondrial compartment specific, since the import of Bcl-2 into the outer membrane was not affected. Contractile activity also modified the mitochondrial content of proteins comprising the import machinery, as evident from increases in the levels of the intramitochondrial chaperone mtHSP70 as well as the outer membrane import receptor Tom20 in SS and IMF mitochondria. Addition of cytosol isolated from stimulated or control muscles to the import reaction resulted in similar twofold increases in the ability of mitochondria to import malate dehydrogenase, despite elevations in the concentration of mitochondrial import-stimulating factor within the cytosol of chronically stimulated muscle. These results suggest that chronic contractile activity modifies the extra- and intramitochondrial environments in a fashion that favors the acceleration of precursor protein import into the matrix of the organelle. This increase in protein import is likely an important adaptation in the overall process of mitochondrial biogenesis.