Direct evidence for the control of mitochondrial respiration by mitochondrial creatine kinase in oxidative muscle cells in situ

The efficiency of stimulation of mitochondrial respiration in permeabilized muscle cells by ADP produced at different intracellular sites, e.g. cytosolic or mitochondrial intermembrane space, was evaluated in wild-type and creatine kinase (CK)-deficient mice. To activate respiration by endogenous production of ADP in permeabilized cells, ATP was added either alone or together with creatine. In cardiac fibers, while ATP alone activated respiration to half of the maximal rate, creatine plus ATP increased the respiratory rate up to its maximum. To find out whether the stimulation by creatine is a consequence of extramitochondrial [ADP] increase, or whether it directly correlates with ADP generation by mitochondrial CK in the mitochondrial intermembrane space, an exogenous ADP-trap system was added to rephosphorylate all cytosolic ADP. Under these conditions, creatine plus ATP still increased the respiration rate by 2.5 times, compared with ATP alone, for the same extramitochondrial [ADP] of 14 microM. Moreover, this stimulatory effect of creatine, observed in wild-type cardiac fibers disappeared in mitochondrial CK deficient, but not in cytosolic CK-deficient muscle. It is concluded that respiration rates can be dissociated from cytosolic [ADP], and ADP generated by mitochondrial CK is an important regulator of oxidative phosphorylation.     

Altered mitochondrial sensitivity for ADP and maintenance of creatine-stimulated respiration in oxidative striated muscles from VDAC1-deficient mice

Voltage-dependent anion channels (VDACs) form the main pathway for metabolites across the mitochondrial outer membrane. The mouse vdac1 gene has been disrupted by gene targeting, and the resulting mutant mice have been examined for defects in muscle physiology. To test the hypothesis that VDAC1 constitutes a pathway for ADP translocation into mitochondria, the apparent mitochondrial sensitivity for ADP (Km(ADP)) and the calculated rate of respiration in the presence of the maximal ADP concentration (Vmax) have been assessed using skinned fibers prepared from two oxidative muscles (ventricle and soleus) and a glycolytic muscle (gastrocnemius) in control and vdac1(-/-) mice. We observed a significant increase in the apparent Km((ADP)) in heart and gastrocnemius, whereas the V(max) remained unchanged in both muscles. In contrast, a significant decrease in both the apparent Km((ADP)) and V(max) was observed in soleus. To test whether VDAC1 is required for creatine stimulation of mitochondrial respiration in oxidative muscles, the apparent Km((ADP)) and Vmax were determined in the presence of 25 mm creatine. The creatine effect on mitochondrial respiration was unchanged in both heart and soleus. These data, together with the significant increase in citrate synthase activity in heart, but not in soleus and gastrocnemius, suggest that distinct metabolic responses to altered mitochondrial outer membrane permeability occur in these different striated muscle types.     

A comparison of in vivo catalysis by creatine kinase in avian skeletal muscles with different fibre composition

The maximum activity of creatine kinase in vitro is similar in the pectoralis major muscle of the chicken and the duck. However, the flux (phosphocreatine to ATP) as measured by 31P saturation transfer NMR in vivo is almost 2-fold higher in the duck. This apparent discrepancy can be accounted for by the differences in the cytosolic free ADP concentrations in resting muscle.     

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.     

Repair of a huge defect of the gluteal region by rotation of a combined tensor fasciae latae-sartorius myocutaneous flap

Although the tensor fasciae latae myocutaneous flap is convenient for covering some defects in the gluteal region, it is not suitable to repair a huge defect because of its limited area. Based on the close relationship of the sartorius and the tensor fasciae latae at their origins and blood supply, the authors designed a myocutaneous flap containing both the tensor fasciae latae and the sartorius muscles and their skin territories with an area exceeding 800 cm2. Two successfully repaired patients are reported. The flaps provide normal sensitivity. The vascular pedicle has a reliable anatomy, being easily dissected, and averages 4.6 to 5.8 cm in length. Both muscles are expendable. There is little functional difficulty for hip joint after the operation.     

Phosphocreatine content of freeze-clamped muscle: influence of creatine kinase inhibition.

The study of cellular energetics is critically dependent on accurate measurement of high-energy phosphates. Muscle values of phosphocreatine (PCr) vary greatly between in vivo measurements (i.e., by nuclear magnetic resonance) and chemical measurements determined from muscles isolated and quick-frozen. The source of this difference has not been experimentally identified. A likely cause is activation of ATPases and phosphotransfer from PCr to ADP. Therefore, rat hindlimb skeletal muscle was perfused either with or without 2 mM iodoacetamide, a creatine kinase inhibitor, and muscle was freeze-clamped either at rest or after contraction. Creatine kinase inhibition resulted in approximately 6 micromol/g higher PCr and lower creatine in the freeze-clamped soleus, red gastrocnemius, and white gastrocnemius. This PCr content difference was reduced when the initial PCr content was decreased with prior contractions. Therefore, the amount of PCr artifact appears to scale with initial PCr content within a fiber-type section. This artifact directly affects the measurement and, thus, the calculations of muscle energetic parameters from studies using isolated and frozen muscle.     

Innervation zones of the upper and lower limb muscles estimated by using multichannel surface EMG

The distribution of innervation zones was investigated in 3 subjects for 17 muscles and 8 muscle groups in the upper and lower limb, by detecting bi-directional propagation of motor unit action potentials (MUAPs) with the multichannel surface electrode array. Clarification of the distribution of innervation zones depended on the ease in detecting the propagation of MUAPs and the actual scattering of innervation zones, which were closely related with muscle morphology with respect to the arrangements of muscle fibers. In muscles having fibers running parallel to each other, such as the biceps brachii, intrinsic hand muscles, vastus lateralis and medialis, tensor fasciae latae, peronei, soleus, tibialis anterior, and hypothenar muscles in the foot, it was relatively easy to detect the propagating MUAPs, and the innervation zones were distributed in a relatively narrow band around muscle belly. On the other hand, in muscles with a complicated structure including pinnation of muscle fibers, in-series muscle fibers and aponeurotic tissues, such as the deltoid, flexors and extensors in the forearm, rectus femoris, sartorius, hamstrings and gastrocnemius, it was more difficult to detect the propagating MUAPs and to identify the innervation zones, which were widely scattered or distributed in complex configurations. The distribution of the innervation zones clarified in the present study can be used to find the optimal location of electrodes in surface EMG recordings and of stimulus electrodes in the functional and therapeutic electrical stimulations. It may also be useful in motor point biopsy for diagnosis of neuromuscular diseases as well as in the botulinum toxin injection for the treatment of spasticity.     

Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice

The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and m. soleus, the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the Km for ADP in the reactions of oxidative phosphorylation. In m. soleus, a low Km for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion, our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux.     

Dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space of rat-heart mitochondria

To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ATP]/[ADP] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ATP]/[ADP] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 +/- 4.7) 10(4) microliters X mg-1 X min-1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 microM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.     

Biochemical adaptation in the skeletal muscle of rats depleted of creatine with the substrate analogue beta-guanidinopropionic acid.

Rats were fed on a diet containing 1% beta-guanidinopropionic acid (GPA), a creatine substrate analogue, for 6-10 weeks to deplete their muscle of creatine. This manipulation was previously shown to give a 90% decrease in [phosphocreatine] in skeletal and cardiac muscle and a 50% decrease in [ATP] in skeletal muscle only. Maximal activities of creatine kinase and of representative enzymes of aerobic and anaerobic energy metabolism were measured in the superficial white, medial and deep red portions of the gastrocnemius muscle, in the soleus and plantaris muscle and in the heart. Fast-twitch muscles were smaller in GPA-fed animals than in controls, but the size of the soleus muscle was unchanged. The activities of aerobic enzymes increased by 30-40% in all fast-twitch muscle regions except the superficial gastrocnemius, but were unchanged in the soleus muscle. The activities of creatine kinase and phosphofructokinase decreased by 20-50% in all skeletal-muscle regions except the deep gastrocnemius, and the activity of glycogen phosphorylase generally paralleled these changes. There were no significant changes in the activities of any of the enzymes measured in the heart. The glycogen content of the gastrocnemius-plantaris complex was increased by 185% in GPA-fed rats. The proportion of Type I fibres in the soleus muscle increased from 81% in control rats to 100% in GPA-fed rats, consistent with a previous report of altered isometric twitch characteristics and a decrease in the maximum velocity of shortening in this muscle [Petrofsky & Fitch (1980) Pflugers Arch. 384, 123-129]. We conclude that fast-twitch muscles adapt by a combination of decreasing diffusion distances, increasing aerobic capacity and decreasing glycolytic potential. Slow-twitch muscles decrease glycolytic potential and become slower, thus decreasing energy demand. These results suggest that persistent changes in the [phosphocreatine] and [ATP] are alone sufficient to alter the expression of enzyme proteins and proteins of the contractile apparatus, and that fibre-type-specific thresholds exist for the transformation response.     

Effect of lipid infusion on metabolism and force of rat skeletal muscles during intense contractions.

The hypothesis that during intense muscle contraction induced by electrical stimulation, long chain fatty acids (LCFA) might reduce mitochondrial ATP/ADP ratio, raising the contribution of glycolysis for ATP production was examined. The effect of a lipid infusion (Lipovenus emulsion) on UCP-3 mRNA level, lactate, glucose-6-phosphate (G-6P) and glycogen content was investigated in rat. Blood samples for determination of free fatty acids and lactate were collected at 0, 30 and 60 min during rest and at 0, 10 and 20 min during muscle contraction. The content of lactate, glycogen and G-6P was also determined in soleus (SO), red gastrocnemius (RG) and white gastrocnemius (WG) muscles collected immediately after muscle contraction period. In addition, the force level was determined during muscle contractions. The effect of Lipovenus emulsion on respiration of mitochondria isolated from rat skeletal muscle, and content of UCP-3 and lactate in cultured skeletal muscle cells was also determined. The in vivo experiments showed that Lipovenus induced a significant increase of UCP-3 mRNA levels. After Lipovenus infusion, lactate level was increased in RG muscle only, whereas the contents of glycogen and G-6P were decreased in both RG and WG muscles (P < 0.05). Lipovenus infusion failed to exert any effect on muscle force performance (P > 0.05). The in vitro experiments showed that Lipovenus infusion induced a significant increase in mitochondrial respiration, but had no effect on UCP-3 content. Lactate concentration was significantly increased in the culture medium of stimulated cells in the control and Lipovenus groups compared with the respective not-stimulated cells (P< 0.05). We concluded that as mitochondrial function becomes limited by the FFA-uncoupling effect, the ATP demand is mainly supplied by anaerobic glucose metabolism preventing an expected decrease in muscle contraction performance.     

Effect of long-term arterial hypotension on the relative volume of functioning capillaries in working skeletal muscles

In control rats, the relative red cell volume, mirroring the number of the functioning capillaries, was substantially greater in the soleus than in the gastrocnemius muscle, while the rise of this parameter seen during contraction of both the muscles, provoked by stimulation of the sciatic nerve, amounted to 37 and 54%, respectively. Fourteen days and 3-4 months after abdominal aorta constriction by a metal spiral, that produced a 30-50% lowering of the blood pressure in the vessels of the animal's body posterior, contractions of the gastrocnemius muscle did not provoke any increase in the relative red cell volume, whereas contractions of the soleus muscle were accompanied by a marked elevation of the relative red cell volume. It is assumed that the mechanisms underlying the working hyperemia of the gastrocnemius and soleus muscles are different.     

The Mechanisms of Post-Tetanic Potentiation in Cat Soleus and Gastrocnemius Muscles

Post-tetanic potentiation of muscle contraction strength (PTP) occurs in cat soleus and gastrocnemius muscles. However, the mechanisms of potentiation are different in these two muscles. Soleus PTP is predominantly a neural event. The application of a high frequency stimulus to the soleus nerve regularly causes each subsequent response to a single stimulus to become repetitive. This post-tetanic repetitive activity (PTR) originates in the motor nerve terminal and is transmitted to the muscle. Consequently each potentiated soleus contraction is a brief tetanus. In gastrocnemius PTR occurs too infrequently to account for PTP. Furthermore, PTP occurs in curarized directly stimulated gastrocnemius muscles to the same extent as in the indirectly stimulated muscle. In this instance PTP is a muscle phenomenon.     

A re-evaluation of conditions required for an accurate estimation of the extramitochondrial ATP/ADP ratio in isolated rat-liver mitochondria

The values reported in the literature for the extramitochondrial ATP/ADP ratio in resting rat-liver mitochondria (State 4) vary widely. The conditions required for an accurate determination of this parameter were therefore investigated. In experiments with rat-liver mitochondria incubated under State-4 conditions, it was found that the extramitochondrial ATP/ADP ratio, as calculated from the values measured in neutralised perchloric acid extracts, was lower than that estimated from the concentrations of creatine and creatine phosphate, using the metabolite indicator method. The discrepancy is due to hydrolysis of ATP occurring in the presence of perchloric acid. Conditions are described for minimising ATP hydrolysis in the presence of perchloric acid, and include the use of low concentrations of perchloric acid, short times of exposure to the acid before neutralisation, low temperatures and the presence of excess EDTA. Under these conditions, the values obtained for the extramitochondrial ATP/ADP ratio agreed with those calculated by the metabolite indicator method, provided ratios do not exceed the value of 100. In cases where the extramitochondrial ATP/ADP does exceed 100, phenol/chloroform/isoamyl alcohol must be used to quench the reactions, as described by Slater et al. (Slater, E.C., Rosing, J. and Mol, A. (1973) Biochim. Biophys. Acta 292, 534-553). With this method, the extramitochondrial ATP/ADP ratio was found to have a value of more than 1000 in rat-liver mitochondria incubated with succinate + rotenone in the resting state (pH 7.0; T = 37 degrees C), in agreement with Slater et al.     

Enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles of hypocaloric rats.

1. The effect of hypocaloric feeding (25% of normal food intake for 21 days) of rats on the enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles was studied. 2. In control and hypocaloric rats the muscle relaxation rates at 100 Hz were 35.76 and 11.38% force loss/10 ms respectively. Control rats exhibited enhanced force of muscle contraction as the frequency of stimulation increased from 10 to 100 Hz, with maximum force being at 100 Hz. Hypocaloric rats exhibited a decrease in the increment of force being exerted at high frequencies, with maintenance of force at lower stimulatory frequencies. 3. In muscles of hypocaloric rats, there were significant decreases in the maximal activities of hexokinase (17.6-37.0%), 6-phosphofructokinase (22.7-34.2%), pyruvate kinase (21.2-36.0%), citrate synthase (34.1-41.5%), oxoglutarate dehydrogenase (29.4-52.4%) and 3-hydroxyacyl-CoA dehydrogenase (26.7-32.1%), whereas the activities of glycogen phosphorylase increased (23.8-43.4%) compared with control values. 4. In soleus-muscle strip preparations of hypocaloric rats, there were significant decreases in the rates of lactate production (28.1%) and glucose oxidation (32.6%) compared with control preparations. 5. Mitochondrial preparations from muscles of hypocaloric rats incubated with various substrates exhibited decreased rates of oxygen uptake compared with control preparations. 6. In muscles of hypocaloric rats (gastrocnemius and soleus), there were significant decreases in the concentrations of glycogen (P less than 0.001) and phosphocreatine (P less than 0.001) and increases in those of pyruvate (P less than 0.001), lactate (P less than 0.001) and ADP (P less than 0.001), whereas those of ATP and AMP remained unchanged. 7. Calculated [lactate]/[pyruvate] and [ATP]/[ADP] ratios exhibited significant increases (P less than 0.05) and decreases (P less than 0.05) in muscles of hypocaloric rats respectively. 8. The results are discussed in relation to the genesis of muscle dysfunction caused by malnutrition.     

Compartmentation of high-energy phosphates in resting and beating heart cells

The subcellular distribution of ATP, ADP, creatine phosphate and creatine has been analyzed by fast detergent fractionation of isolated frog heart cells. Digitonin fractionation (0.5 mg/ml, 10 s at 2 degrees C in 20 mM 4-morpholinepropanesulfonic acid/3 mM EDTA/230 mM mannitol medium) was used to separate mitochondria and myofilaments from cytosol. To separate myofilaments from the other cellular compartments. Triton X-100 was used (2%, 15 s in the same medium as digitonin). For either resting or beating cells the total cellular contents of ATP, ADP, creatine phosphate and creatine was similar, nevertheless the O2 consumption was 6-times higher. The compartmentation of these metabolites was also identical. Myofilaments contain 1.1 nmol ADP per mg total cellular proteins. In the cytosolic compartment the metabolite concentrations, all measured in nmol per mg total cellular proteins, were: ATP, 13; ADP, 0.25-0.05; creatine phosphate, 18.5 and creatine, 14. This indicated that the reaction catalyzed by creatine kinase was in a state of (or near) equilibrium.     

Some properties of different skeletal muscle fiber types: comparison of reference bases

Several biochemical components of the white portion of the gastrocnemius (WGM), plantaris (PM), and soleus (SM) muscles of the rat and middle gluteal (MGM) muscle of the horse were compared based on wet and dry weight, protein, and total creatine concentrations ([TCr]). The water content was similar for the rat hindlimb muscles, however, the concentrations of protein, ATP, phosphocreatine (PCr), creatine, and glycogen ranked as SM less than PM less than WGM for all reference bases except total creatine. In contrast, concentrations of ATP, creatine, and PCr were similar in all muscles studied when expressed as ratios of [TCr]. Horse MGM had the lowest percent of water and protein per gram wet or dry weight but highest glycogen concentration of the muscles studied, irrespective of the reference base used to express concentrations. Coefficients of variation were lowest when muscle constituents were related to [TCr]. It is concluded that expressing muscle constituents relative to total creatine results in the smallest variation and is a good method for making comparisons between muscles of similar fiber composition. However, essential information concerning different types of muscle may be lost when this reference base is used.     

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue beta-guanidinopropionic acid

Rats were fed a diet containing 1% beta-guanidinopropionic acid (GPA) for 6-10 weeks to deplete their skeletal muscle of creatine. 31P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 mumol X g-1) and the [ATP] to 50% (3.3 mumol X g-1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 mumol X g-1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM X s-1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM X s-1). The rate of PGPA resynthesis during recovery (0.018 mM X s-1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM X s-1). The creatine kinase flux (creatine phosphate----ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM X s-1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.     

The vascular supply of the extended tensor fasciae latae flap: how far can the skin paddle extend?

The vascular supply of the tensor fasciae latae flap and of the lateral thigh skin was studied in 10 cadavers to evaluate whether the lateral thigh skin toward the knee could be incorporated into an extended tensor fasciae latae flap. Within each cadaver, vascular injection of radiopaque material preceded flap elevation in one limb and followed flap elevation in the contralateral limb. Flaps raised after vascular injection were examined radiographically to evaluate the vascular anatomy of the lateral thigh skin independent of flap elevation. When vascular injection was made into the profunda femoris, the upper two-thirds of the flaps was better visualized than the distal third. When the injection was made into the popliteal artery, the vasculature of the distal third of the flaps was better visualized. Flaps raised before vascular injection were examined radiographically to delineate the anatomical territory of the vascular pedicle that had been injected. In these flaps, consistent cutaneous vascular supply was only seen in the skin overlying the tensor fasciae latae muscle, confirming that musculocutaneous perforators are the predominant means by which the pedicle of the tensor fasciae latae flap supplies the skin of the lateral thigh. Extended tensor fasciae latae flaps were elevated bilaterally in one cadaver, and selective methylene blue injections were made into the lateral circumflex femoral artery on one side and into the superior lateral genicular artery on the contralateral side. Methylene blue was observed in the proximal and distal thirds of the skin paddles, respectively, leaving unstained midzones. The vascular network of the lateral thigh skin could be divided into three zones. The lateral circumflex femoral artery and the third perforating branches of the profunda femoris artery perfuse the proximal and middle zones of the lateral thigh skin, respectively. The superior lateral genicular artery branch of the popliteal artery perfuses the distal zone. The middle and distal zones meet 8 to 10 cm above the knee joint, where the skin paddle of the tensor fasciae latae flap becomes unreliable. These data indicate that if the aim is to incorporate the skin over the distal thigh in an extended tensor fasciae latae flap without resorting to free-tissue transfer, then either a carefully planned delay procedure or an additional anastomosis to the superior lateral genicular artery is required.