Subsarcolemmal Research Articles

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.

Regional differences in spontaneous Ca2+ spark activity and regulation in cat atrial myocytes

Calcium sparks result from the concerted opening of a small number of Ca2+ release channels (ryanodine receptors, RyRs) organized in clusters in the membrane of the sarcoplasmic reticulum (SR). Calcium sparks represent the elementary events of SR Ca2+ release in cardiac myocytes, and their spatial and temporal summation results in whole-cell [Ca2+]i transients observed during excitation-contraction coupling (ECC). Atrial myocytes generally lack transverse tubules; however, during ECC Ca2+ release is initiated from junctional SR (j-SR) in the cell periphery from where activation propagates inwardly through Ca(2+)-induced Ca2+ release (CICR) from non-junctional SR (nj-SR). Despite the structural differences in the microdomains of RyRs of j-SR and nj-SR, spontaneous Ca2+ sparks are observed from both types of SR, albeit at different frequencies. In cells that showed spontaneous Ca2+ sparks from j-SR and nj-SR, subsarcolemmal (SS) Ca2+ sparks from the j-SR were 3-4 times more frequent than central (CTR) Ca2+ sparks occurring from nj-SR. Subsarcolemmal Ca2+ sparks had a slightly higher amplitude, but were essentially identical in their spatial spread and duration when compared to CTR Ca2+ sparks. Sensitization of RyRs with a low concentration (0.1 mM) of caffeine led to a 107% increase in the frequency of CTR Ca2+ sparks, whereas the SS Ca2+ spark frequency increased by only 58%, suggesting that the nj-SR is capable of much higher Ca2+ spark activity than observed normally in unstimulated cells. The L-type Ca2+ channel blocker verapamil reduced SS Ca2+ spark frequency to 38% of control values, whereas Ca2+ spark activity from nj-SR was reduced by only 19%, suggesting that SS Ca2+ sparks are under the control of Ca2+ influx from the extracellular space. Removal of extracellular Ca2+ eliminated SS Ca2+ sparks completely, whereas Ca2+ sparks from the nj-SR continued, albeit at a lower frequency. In membrane-permeabilized (saponin-treated) atrial myocytes, where [Ca2+] can be experimentally controlled throughout the entire myocyte, j-SR and nj-SR Ca2+ spark frequencies were identical, and Ca2+ sparks could be observed spaced at sarcomeric distances throughout the entire cell, suggesting that all release sites of the nj-SR can become active. Measurement of SR Ca2+ load (10 mM caffeine) revealed no difference between j-SR and nj-SR. The data suggest that in atrial myocytes, which lack a t-tubular system, the nj-SR is fully equipped with a three-dimensional array of functional SR Ca2+ release sites; however, in intact cells under resting conditions, peripheral RyR clusters have a higher probability of activation owing to their association with surface membrane Ca2+ channels, leading to higher spontaneous Ca2+ spark activity. In conclusion, Ca2+ sparks originating from both j-SR and nj-SR are rather stereotypical and show little differences in their spatiotemporal properties. In intact cells, however, the higher frequency of spontaneous SS Ca2+ sparks arises from the structural arrangement of sarcolemma and j-SR membrane and thus from the difference in the trigger mechanism.

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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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.

Endurance and Interval Training and Palmitoyl L-Carnitine Oxidation by Mitochondria from VLCAD Deficient Mice

2258 Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a fatty acid oxidation disorder that limits oxidation of fatty acids sixteen carbons and longer. However, even in VLCAD deficiency, some residual enzyme activity toward palmitate remains because of the overlap of specificity with LCAD. PURPOSE: This project examined the hypothesis that exercise training would enhance gastrocnemius subsarcolemmal (SS) and intermyoflbrillar (IMF) mitochondrial oxidation of palmitoyl l-carnitine plus malate and that high-intensity interval training would have a greater effect than endurance training. METHODS: The groups were sedentary control mice (C), endurance trained mice (E), and interval trained mice (I). Both groups of training mice exercised 5 dfiwk−1. During the first four weeks of training the durations and intensity of exercise were progressively increased. At the end of the four weeks, endurance training mice were running on the motor driven treadmill at a speed of 24 m fimin−1 for 60 min. Interval training mice ran for 2 min at 24 m fimin−1 followed by 2 min at 41 m. min−1. The intervals were then alternated for a total of 46 min. day−1. Beginning at the end of the 8th week, the gastrocnemius muscle was removed and the oxidation of palmitoyl-carnitine plus malate determined polarographically. RESULTS: Oxidation was significantly (p <0.05) depressed in IMF mitochondria from VLCAD deficient mice but not subsarcolemmal mitochondria. High-intensity interval training caused large increases in palmitoyl l-carnitine oxidation by both subsarcolemmal (p < 0.05) and intermyofibrillar mitochondria. Endurance training did not cause a significant increase in either subsarcolemmal or intermyofibrillar oxidation rates. CONCLUSIONS: Unlike previous observations in mice, these results show that high intensity, interval training is more effective than endurance training in improving fatty acid oxidation, at least in subsarcolemmal mitochondria. Further, the results show that mitochondrial oxidation of palmitoyl l-carnitine is reduced in VLCAD deficient mice relative to non-deficient mice.

Endurance and Interval Training and Palmitoyl L-Carnitine Oxidation by Mitochondria from VLCAD Deficient Mice

2258 Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a fatty acid oxidation disorder that limits oxidation of fatty acids sixteen carbons and longer. However, even in VLCAD deficiency, some residual enzyme activity toward palmitate remains because of the overlap of specificity with LCAD. PURPOSE: This project examined the hypothesis that exercise training would enhance gastrocnemius subsarcolemmal (SS) and intermyoflbrillar (IMF) mitochondrial oxidation of palmitoyl l-carnitine plus malate and that high-intensity interval training would have a greater effect than endurance training. METHODS: The groups were sedentary control mice (C), endurance trained mice (E), and interval trained mice (I). Both groups of training mice exercised 5 dfiwk−1. During the first four weeks of training the durations and intensity of exercise were progressively increased. At the end of the four weeks, endurance training mice were running on the motor driven treadmill at a speed of 24 m fimin−1 for 60 min. Interval training mice ran for 2 min at 24 m fimin−1 followed by 2 min at 41 m. min−1. The intervals were then alternated for a total of 46 min. day−1. Beginning at the end of the 8th week, the gastrocnemius muscle was removed and the oxidation of palmitoyl-carnitine plus malate determined polarographically. RESULTS: Oxidation was significantly (p <0.05) depressed in IMF mitochondria from VLCAD deficient mice but not subsarcolemmal mitochondria. High-intensity interval training caused large increases in palmitoyl l-carnitine oxidation by both subsarcolemmal (p < 0.05) and intermyofibrillar mitochondria. Endurance training did not cause a significant increase in either subsarcolemmal or intermyofibrillar oxidation rates. CONCLUSIONS: Unlike previous observations in mice, these results show that high intensity, interval training is more effective than endurance training in improving fatty acid oxidation, at least in subsarcolemmal mitochondria. Further, the results show that mitochondrial oxidation of palmitoyl l-carnitine is reduced in VLCAD deficient mice relative to non-deficient mice.

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.

Regulation of junctional and non-junctional sarcoplasmic reticulum calcium release in excitation-contraction coupling in cat atrial myocytes.

We have characterized the dependence on membrane potential (V(m)) and calcium current (I(Ca)) of calcium-induced calcium release (CICR) from the junctional-SR (j-SR, in the subsarcolemmal (SS) space) and non-junctional-SR (nj-SR, in the central (CT) region of the cell) of cat atrial myocytes using whole-cell voltage-clamp together with spatially resolved laser-scanning confocal microscopy. Subsarcolemmal and central [Ca(2+)](i) transient amplitudes and I(Ca) had a bell-shaped dependence on V(m), but [Ca(2+)](i) reached a maximum at more negative V(m) (-10 to 0 mV) than I(Ca) (+10 mV). Termination of I(Ca) after a brief depolarization (2.5 to 22.5 ms) immediately interrupted only the SS [Ca(2+)](i) transient, leaving the development of the CT [Ca(2+)](i) transient unaffected. Block of SR function with 20 microM ryanodine and 2 microM thapsigargin, revealed that > 90 % of the control [Ca(2+)](i) transient amplitude was attributable to active SR Ca(2+) release through ryanodine receptors (RyRs). The gain of SR Ca(2+) release was highest in the SS space at negative test potentials and was less pronounced in the CT region. Inhibition of Na(+)-Ca(2+) exchange resulted in prolonged and higher amplitude [Ca(2+)](i) transients, elevated resting [Ca(2+)](i), accelerated propagation of CICR, decreased extrusion of Ca(2+) and an increase in j-SR Ca(2+) load. Increasing the cytosolic Ca(2+) buffer capacity by internal perfusion with 1 mM EGTA limited SR Ca(2+) release to the SS region, indicating that Ca(2+) release from nj-SR is initiated by diffusion of Ca(2+) from the cell periphery and propagating CICR. Junctional-SR Ca(2+) release occurred at discrete sites whose order of activation and amplitude of release varied from beat to beat. In conclusion, during normal excitation-contraction coupling in cat atrial myocytes, only Ca(2+) release from the j-SR is directly activated by Ca(2+) entering via I(Ca). Elevation of SS [Ca(2+)](i) is required to provide the cytosolic Ca(2+) gradient needed to initiate regenerative and propagating CICR from nj-SR.

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.

Morphological effects of an anabolic steroid on muscle fibres of the diaphragm in mice.

Until recently, rats have been used for studies on the effects of anabolic steroids (AS) for improving physical performance. However, no consistent results on the changes in muscle fibre volume have ever been obtained. In this study we investigated the morphological effects of nandrolone phenylpropionate, an AS, on the muscle fibres of the diaphragm in adult male mice. AS (5 mg kg(-1) week(-1)) was injected intramuscularly in the experimental group (AS-administration group), and peanut oil (0.08 ml week(-1)) was injected in the control group. The cross-sectional areas of Type I (red) and Type II (white and intermediate) muscle fibres after 4 weeks of AS administration were 135% and 139% larger than those in the control group, respectively. The mean cross-sectional areas of mitochondria in the subsarcolemmal (SS) region and interfibrillar (IF) region in Type I fibres and the SS region of the Type II muscle fibres in the AS-administration group were 139%, 135% and 124% larger than those in the control group, respectively. No significant differences in the cross-sectional area of mitochondria were noted between the group of mice administered with AS for 8 weeks and the control group, showing that the effect of the AS administration attained a peak at 4 weeks, but that the effect of the 8 weeks of AS administration declined. We concluded from these results that the response of Type I muscle fibres to AS is stronger than that of Type II muscle fibres, as is the response of SS mitochondria compared to IF mitochondria. We also suggested that the two types of mitochondria may have different roles and that administration of AS may cause a different response in the two types of mitochondria.

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.

The effect of aerobic exercise training on the distribution of succinate dehydrogenase activity throughout muscle fibres.

We evaluated the effect of endurance training (cycling 3 times per week for 12 weeks) on succinate dehydrogenase (SDH) activity in the subsarcolemmal (SS) and intermyofibrillar (IMF) regions of vastus lateralis muscle fibres in 7 individuals (4 females and 3 males). SDH activity of the SS region increased 9.4% and 12.8% in type I and II fibres, respectively (p < .05). SDH activity of the IMF region increased 4.7% and 6.7% in type I and II fibres, respectively (p < .05). This was less than the increase in the SS region (p < .05). No significant changes were observed in a control group (4 females and 3 males). These data suggest that mitochondria in the SS and IMF regions of human vastus lateralis muscle fibres are sensitive to endurance training. The greater response in the SS region suggests that the metabolic requirements of SS mitochondria were stressed to a greater extent than IMF mitochondria with endurance training.

Effects of 4 wk of hindlimb suspension on skeletal muscle mitochondrial respiration in rats.

We investigated in rats the effect of 4 wk of hypodynamia on the respiration of mitochondria isolated from four distinct muscles [soleus, extensor digitorum longus, tibial anterior, and gastrocnemius (Gas)] and from subsarcolemmal (SS) and intermyofibrillar (IMF) regions of mixed hindlimb muscles that mainly contained the four cited muscles. With pyruvate plus malate as respiratory substrate, 4 wk of hindlimb suspension produced an 18% decrease in state 3 respiration for IMF mitochondria compared with those in the control group (P < 0.05). The SS mitochondria state 3 were not significantly changed. Concerning the four single muscles, the mitochondrial respiration was significantly decreased in the Gas muscle, which showed a 59% decrease in state 3 with pyruvate + malate (P < 0.05). The other muscles presented no significant decrease in respiratory rate in comparison with the control group. With succinate + rotenone, there was no significant difference in the respiratory rate compared with the respective control group, whatever the mitochondrial origin (SS, or IMF, or from single muscle). We conclude that 4 wk of hindlimb suspension alters the respiration of IMF mitochondria in hindlimb skeletal muscles and seems to act negatively on complex I of the electron-transport chain or prior sites. The muscle mitochondria most affected are those isolated from the Gas muscle.

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 < 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 < 0.05). Also, the increases associated with the IMF mitochondria were greater than the SS mitochondria (p < 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.