Dietary fatty acids affect structural membrane lipid composition, which in turn is strongly linked to cellular function(Reference Gorjao, Azevedo-Martins and Rodrigues1), including myocardial cell function(Reference Siddiqui, Harvey and Zaloga2). Although tissue fatty acid composition reflects the types of fatty acids in the diet(Reference McLennan, Howe and Abeywardena3–Reference Charnock, Abeywardena and Poletti6), patterns of incorporation vary according to the tissue. In particular, heart myocardial membranes incorporate the n-3 PUFA DHA, from the diet, but do so in concentrations well above the circulating concentrations(Reference Charnock, Abeywardena and Poletti6). Although less studied, skeletal muscle appears to share this property of membrane sensitivity to incorporation of DHA with fish oil diet intervention(Reference Charnock, Abeywardena and Poletti6–Reference Ayre and Hulbert8). Furthermore, skeletal muscle membrane composition is influenced by exercise training(Reference Helge, Ayre and Hulbert9–Reference Andersson, Sjodin and Olsson11). In skeletal muscle, the incorporation of membrane n-3 PUFA may even be fibre-type specific and possibly be related to the high oxidative capacity of type I and IIa muscle cells where high levels of PUFA have been measured(Reference Kriketos, Pan and Sutton12, Reference Pan, Lillioja and Milner13).
There is extensive evidence that incorporation of dietary n-3 PUFA into myocardial membranes has cellular(Reference Siddiqui, Harvey and Zaloga2) and direct effects on heart function. Fish oil feeding to increase dietary n-3 PUFA has been demonstrated to enhance mechanical performance of the heart in the marmoset monkey(Reference McLennan, Barnden and Abeywardena14, Reference Charnock, McLennan and McIntosh15) and rat(Reference Abdukeyum, Owen and McLennan16, Reference Pepe and McLennan17), decrease heart rate in exercising horses(Reference O'Connor, Lawrence and Lawrence18) and in rats(Reference Pepe and McLennan5), stabilise membranes to reduce arrhythmia vulnerability in rats(Reference McLennan, Abeywardena and Charnock4) and marmosets(Reference McLennan, Bridle and Abeywardena19) and modulate myocardial O2 consumption in rats(Reference Pepe and McLennan17). In humans, the direct effects of dietary fish oil in the myocardium include reduced fatal arrhythmia(Reference Burr, Gilbert and Holliday20) and reduced resting(Reference Dallongeville, Yarnell and Ducimetiere21, Reference Shah, Ichiuji and Han22) and exercising heart rate(Reference Peoples, McLennan and Howe23). The small but consistent reduction in heart rate has also been distilled from many clinical trials through meta-analysis(Reference Mozaffarian, Geelen and Brouwer24) and indirectly supports the notion that myocardial O2 consumption is reduced when fish is in the diet. Furthermore, there is evidence for reduction in whole body O2 consumption in well-trained cyclists following dietary fish oil intervention(Reference Peoples, McLennan and Howe23), perhaps reflecting lower O2 consumption by the working skeletal muscle. Reduced O2 demand might produce fatigue resistance but study of isolated muscle has produced contradictory results and none have measured O2 consumption. In view of limitations in O2 delivery to isolated muscle bundles, an autologous, blood-perfused hindlimb method was developed(Reference Hoy, Peoples and McLennan25) to investigate the effects of dietary n-3 PUFA on the skeletal muscle O2 consumption and contractile function, measuring both short-term fatigue during repeated contractions and twitch contractile recovery between contraction bouts. The premise of the present study was the confirmed changes in skeletal muscle membrane fatty acid composition through dietary intervention would directly modify skeletal muscle O2 consumption parallel to changes previously reported for heart, which may be expressed as resistance to muscle fatigue.
Methods
Eighteen male Wistar rats (400–500 g) were used for the study (six per dietary intervention). Experiments were assessed and approved by the Animal Ethics Committee from the University of Wollongong, and all national and institutional guidelines were followed. Animals were housed two per cage in the University of Wollongong's animal facility with a room temperature maintained at 23–25°C and a 12 h light–dark cycle.
Diet composition
Three diets were prepared for the present study: saturated fat (SF); n-6 PUFA; n-3 PUFA. The diets all contained 10 % fat by weight. The proportions of fat sources were selected to deliver similar total PUFA in the n-6 PUFA and n-3 PUFA diets, similar n-6 PUFA in the n-3 PUFA and SF diets and similar n-3 PUFA in the n-6 PUFA and SF diets. The final composition of each diet was established using GC against the known standards (Table 1). Olive oil was provided as light (refined) olive oil, therefore free of confounding sources of natural antioxidants; SF as beef tallow; n-6 PUFA as safflower oil; n-3 PUFA as high-DHA tuna fish oil (28·9 % DHA and 9 % EPA) (Clover Corporation, Altona North, VIC, Australia). Diets were prepared from purified ingredients and stored at − 20°C(Reference Owen, Peter-Przyborowska and Hoy26).
Fat sources and concentration of major fatty acids of experimental diets (g/kg diet)*
SF, saturated fat; P/S ratio, ratio of PUFA/SFA; n-6:n-3 ratio, ratio of n-6 PUFA to n-3 PUFA; UI, unsaturated index; ND, not detectable.
* Fatty acid concentrations as determined by GC.
Study design
Animals were fed a wash-out diet containing olive oil for 14 d to standardise muscle membrane fatty acid composition. They were then randomly allocated into three groups and fed ad libitum the SF, safflower oil (n-6 PUFA) or the tuna fish oil (n-3 PUFA) diet for 8 weeks. Fresh food was provided twice per week and daily consumption (g) was estimated by weighing the remainder from the previous feeding. Animal weight was measured once a week.
Surgical preparation for ventilation, muscle perfusion and stimulation
The autologous pump-perfused hindlimb has been previously described in both dogs(Reference Hogan, Gladden and Grassi27) and rats(Reference Hoy, Peoples and McLennan25). In brief, rats (18–20 weeks) were anaesthetised (sodium pentobarbital 6 mg/100 g intraperitoneally) with supplementary injections (2 mg/100 intraperitoneally) as required. Experiments were performed with the rat and perfusion system enclosed in a heated perspex chamber (32·2 ± 0·3°C) with animal body temperature maintained at 37°C (rectal temperature). The rats were prepared for artificial ventilation and blood pressure recording by cannulation of the trachea and left carotid artery, respectively. Rats were ventilated (1 ml/100 g body weight) to ensure constant high arterial O2 (Rodent Ventilator 7025, Ugo Basile, Italy).
All cannulae were fluid filled with saline containing 6 % dextran (w/v) and 5000 IU heparin/100 ml (Sigma-Aldrich, Castle Hill, NSW, Australia). Arterial (oxygenated) blood was accessed via a cannula inserted into the right femoral artery (non-perfused leg) towards the heart and was connected to a re-sealing flexi-tube, passed through a peristaltic roller pump (Gilson Minipuls 3, Middleton, WI, USA). Arterial blood was then passed through a cannula that was inserted into the left femoral artery (perfused leg) and positioned to deliver blood directly to muscle groups below the knee. A T-junction inserted in the blood flow line on the perfused hindlimb side of the pump was connected to a pressure transducer (Argon CDXIII; Maxim Medical, Athens, TX, USA) for measurement of hindlimb perfusion pressure. Venous blood (de-oxygenated) return to the heart and lungs was achieved via cannula inserted into the left femoral vein of the perfused leg and right jugular vein at the other end for passive flow.
Hindlimb muscle contraction was achieved via sciatic nerve stimulation. A bipolar electrode (F-B5EI; Grass Instrument Division, West Warwick, RI, USA) was placed under the nerves for direct electrical stimulation of the trunk supplying the muscles of the gastrocnemius–plantaris–soleus muscle bundle. The nerve was crushed above the stimulation point to prevent retrograde conduction. The gastrocnemius–plantaris–soleus muscle group tendons were tied with non-compliant silk and connected to a force transducer (FT03C; Grass Instrument Division). Saline-soaked gauze was placed over the muscles to prevent them from drying.
Hindlimb perfusion and stimulation protocol
All hindlimbs were perfused for 30 min at 1 ml/min to allow for perfusion pressure to reach steady state (approximately 100 mmHg). Baseline arterial and venous blood samples (400 μl) were drawn after 30 min of the initial perfusion (flow = 1 ml/min) with no stimulation. Three separate 10 min stimulation bouts (1 Hz, 7–12 V, 0·05 ms) were delivered to the sciatic nerve separated by 30 min of muscle recovery. The flow rate was 1·5 ml/min during contraction periods and returned to 1 ml/min during the recovery periods. Arterial and venous samples were collected between the ninth and tenth minute of each contraction bout and between 25 and 30 min of recovery between bouts.
Measurements
Blood pressure and twitch force
Data were referenced to ground and amplified (Onspot Australia, Barrack Heights, NSW, Australia). The data acquisition software, Labview for Windows (National Instruments, Austin, TX, USA), was used to collect both pressure and twitch force simultaneously during the perfusion trials at a sampling rate of 200 Hz.
Blood samples
Arterial and venous blood samples (400 μl) were collected via the re-sealing silicone sections of the cannulae located proximal and distal to the hindlimb. Of the 400 μl blood sampled, 240 μl was presented to the laboratory blood gas and electrolyte machine (BGElectrolytes; Instrumentation Laboratory, Lexington, MA, USA) for measurement of PO2, PCO2, Na+, K+, pH and Hb. The remaining 150–200 μl was spun down on a bench-top micro centrifuge (Milipore, North Ryde, NSW, Australia), the plasma was removed and frozen for later analysis. Erythrocytes from the plasma collections were re-suspended in an equal volume of saline and re-injected into the venous side of the perfusion. Hb levels were maintained above 12·5 g/100 ml whole blood.
Muscle samples
Control muscle samples (gastrocnemius, plantaris and soleus) were taken from the right (non-perfused) leg immediately before the right femoral artery cannulation, to be used as within-animal control tissue. At the completion of each experiment and while the blood was still flowing, left leg (perfused and contracted) muscle samples (gastrocnemius, plantaris and soleus) were rapidly extracted. All muscles were freeze-clamped and stored ( − 80°C) for later analysis. Soleus was used for red/oxidative tissue and gastrocnemius portion was used for white/glycolytic tissue. Additional muscles (deep posterior tibial, extensor digitorum longus and tibialis anterior) from the perfused hindlimb were collected to establish total weight of the muscle involved in the perfusion.
Skeletal muscle membrane fatty acid analysis
The method of membrane fatty acid analysis has been described previously(Reference Pan and Storlien7). In brief, 100–150 mg of skeletal muscle tissue were weighed and homogenised in a chloroform–methanol mixture (2:1, v/v) (analytical grade Fluka; Sigma-Aldrich). Steps were taken for total lipid extraction using 1 m-H2SO4 (Sigma-Aldrich), phospholipid separation via silica seppack columns (Waters, Sutton, MA, USA), methylation of phospholipid fatty acids using 14 % boron trifluoride in methanol ((Sigma-Aldrich) stored at 0–4°C) heated at 85°C for 1 h, purification via florisil Seppack columns (Waters) using diethyl ether (5 %) (Fluka; Sigma-Aldrich) in petroleum spirit (7 ml) (Fluka; Sigma-Aldrich) and finally GC (Shimadzu GC-17A, 30 m × 0·25 mm internal diameter capillary column, total run time 23 min). All solvents were freshly prepared at the time of analysis and contained 0·01 % (w/v) butylated hydroxy toluene (Sigma-Aldrich). Individual fatty acids were identified by comparison with the known standards in the laboratory. The relative amount of each fatty acid was determined by integrating the area under the peak and dividing by the result for all fatty acids detected. Unsaturation index, which represents the number of double bonds per hundred fatty acid molecules, was calculated by summing the products of the proportion (mol%) of each fatty acid multiplied by the number of double bonds.
Statistical analysis
Computer software, Statistix, version 8 (Tallahassee, FL, USA), was used to analyse the data. One- (diet) and two-way (diet and time) ANOVA with individual means were compared using a corrected Bonferronni's post hoc analysis to indicate the significant difference. α was set at P < 0·05. Data were expressed as means with their standard errors.
Results
Food consumption and body weight
There was no significant difference in the quantity of food consumed between dietary groups at either the start, the end or as a mean weekly intake over the 8-week feeding period (SF: 6·85 (sem 0·12); n-6 PUFA: 6·68 (sem 0·15); n-3 PUFA: 6·70 (sem 0·11) g/100 g body weight per week). Mean body weight was lower in the n-6 PUFA group (SF: 491 (sem 27); n-6 PUFA: 427 (sem 9); n-3 PUFA: 479 (sem 15) g (P < 0·05 n-6) PUFA v. SF and n-3 PUFA). Despite the differences in mean body weight, there were no significant differences between dietary groups in mean total hindlimb muscle (SF: 4·48 (sem 0·14); (n-6) PUFA: 4·23 (sem 0·17); n-3 PUFA: 4·36 (sem 0·15) g wet weight) or gastrocnenius–plataris–soleus muscle bundle (SF: 2·42 (sem 0·19); n-6 PUFA: 2·34 (sem 0·11); (n-3) PUFA: 2·20 (sem 0·11) g wet weight).
Skeletal muscle membrane fatty acids
The fish oil fed group showed several marked membrane fatty acid concentration differences compared with the SF and n-6 PUFA groups, which did not differ from each other in any significant way (Table 2). In red and white portions of skeletal muscle, the n-3 PUFA group had significantly lower 18 : 2n-6c (linoleic acid), 20 : 4n-6 (arachidonic acid) and total n-6 PUFA (P < 0·05) and significantly higher 20 : 5n-3 (EPA), 22 : 6n-3 (DHA) (Fig. 1(A), P < 0·05), and total n-3 PUFA (Fig. 1(B), P < 0·05) compared with both the SF and (n-6) PUFA groups. This was reflected by the significantly lower n-6:n-3 PUFA ratio for the n-3 PUFA muscles (Table 2, P < 0·05). There were no significant differences that could be attributed to the red (soleus) or white (gastrocnemius) portion of muscle for these diets. The unsaturation index was significantly elevated in n-3 PUFA muscles compared with the SF and n-6 PUFA groups, which were not different from each other. There were no significant differences between groups in either total SFA or total PUFA.
(Mean values with their standard errors)
SF, saturated fat; ΣSFA, sum of SFA; ΣPUFA, sum of PUFA; Σ(n-6), sum of (n-6) PUFA; Σ(n-3), sum of (n-3) PUFA; UI, unsaturation index; n-6:n-3, ratio of n-6 PUFA to n-3 PUFA; nd, not detectable.
a,b Mean values within a row with unlike superscript letters were significantly different (P < 0·05).
* Fatty acid concentrations as determined by GC.
Skeletal muscle PUFA phospholipid concentrations for saturated fat (SF), safflower oil (n-6 PUFA) and DHA-rich fish oil (n-3 PUFA) after 8 weeks feeding. (A) Total n-3 PUFA; (B) DHA concentration. Mean values with unlike letters between diet groups were significantly different (P < 0·05). Mean values with their standard errors. □, SF; 
, n-6 PUFA; ■, n-3 PUFA.
Baseline perfusion
There was no significant difference in rectal temperature between dietary groups monitored during the procedure (SF: 37·0 (sem 0·9); n-6 PUFA: 36·7 (sem 0·6); n-3 PUFA: 36·0 ± 0·7°C). Hindlimb perfusion pressure after 30 min (1 ml/min) was significantly higher in the SF group compared with the n-3 PUFA group (SF: 126 (sem 10); n-6 PUFA: 102 (sem 14); n-3 PUFA: 93 (sem 10) mmHg) (P < 0·05). There were no significant differences between dietary groups in the baseline arterial blood samples (Table 3). In all groups, artificial ventilation and resulting PaO2 were sufficient to achieve approximately 98 % O2 saturation of the erythrocytes and corresponding high content of arterial O2 supply (approximately 19–20 ml/100 ml). Baseline venous samples demonstrated a significant drop in PO2 compared with arterial blood (P < 0·05) and uptake of O2 across the resting muscle bundle. Although NS, the n-3 PUFA group demonstrated a trend for lower (a − v)O2 difference (ml/100 per ml) and basal O2 consumption (μmol/g per min) (Table 3) compared with both the n-6 PUFA (P = 0·13) and SF (P = 0·09) groups.
(Mean values with their standard errors)
SF, saturated fat.
a,b Mean values within a row with unlike superscript letters were significantly different (P < 0·05).
Muscle contraction
There was a significant decline in initial and maximum tensions between Bout 1 and Bout 3 independent of diet (P < 0·05) (Fig. 2). The decline was smallest in the n-3 PUFA group for both initial and maximum tension (P = 0·07 and 0·09, respectively). There was no significant difference in the final tension between Bout 1 and Bout 3 independent of diet; however, final tension in the n-3 PUFA group was significantly higher compared with the SF group (P < 0·05). As hindlimb blood flow increased, perfusion pressure significantly increased on each occasion independent of dietary group (P < 0·05). There were no significant effects of diet on hindlimb perfusion pressure during twitch contractions.
Twitch tension development (N/g wet weight) for Bout 1, Bout 2 and Bout 3 for (A) saturated fat (SF), –□–, Bout 1; - -□- -, Bout 2; 
, Bout 3; (B) n-6 PUFA (safflower oil), –▿–, Bout 1; - -▿- -, Bout 2; …▿…, Bout 3 and (C) n-3 PUFA (DHA-rich fish oil) groups, –○–, Bout 1; - -○- -, Bout 2; …○…, Bout 3. Flow = 1·5 ml/min. Initial tension (time = 0), maximum tension (time = 60 s) and final tension (time = 10 min) are shown (n 6). Values displayed as means with their standard errors.
The recovery of initial, maximum and final twitch tension in Bout 3 (1·5 ml/min) was compared to Bout 1 (1 ml/min) (Fig. 3(A)). The n-3 PUFA group demonstrated a greater recovery of twitch tension in contraction Bout 3 (P < 0·05) compared with the SF and n-6 PUFA groups. Additionally, when final twitch tension was expressed as a percentage of maximum twitch tension (Fig. 3(B)), in bouts 1, 2 and 3, the n-3 PUFA group demonstrated greater maintenance of twitch tension over the 10 min (P < 0·05). Twitch characteristics including maximum rate of contraction (+dT/dt) and maximum rate of relaxation ( − dT/dt) decreased across Bouts 1, 2 and 3 independent of diet. There was no significant difference between diets at the points of initial, maximum and final twitch tension (data not shown).
(A) Recovery (%) for initial, maximum and final twitsh tension Bout 3 v. Bout 1. (B) Final twitch tension as a percentage of maximum twitch tension (Bout 1, Bout 2 and Bout 3). □, SF; , n-6 PUFA; ■, n-3 PUFA. Mean values with unlike superscript letters between diet groups were significantly different (P < 0·05) (n 6). Values displayed as mean with their standard errors.
During contraction, the (a − v)O2 difference (ml/100 per ml) and O2 consumption (μmol/g per min) (Fig. 4(B)) were significantly lower in the n-3 PUFA group compared with the SF group. The efficiency index, calculated as the twitch tension developed relative to muscle mass for a given amount of O2 consumed (N/g per μmol per min), was significantly higher in the n-3 PUFA group across all exercise bouts compared with both SF and n-6 PUFA groups (P < 0·05) (Fig. 4(C)).
(A) Oxygen consumption (μmol/g × min) at baseline and recovery 1 and recovery 2 (following Bout 1 and 2 respectively). Flow rate = 1 ml/min, (B) contracting O2 consumption (μmol/g × min) and (C) efficiency index (N/100 g per µmol × min) at flow rate 1.5 ml/min (Bout 1, Bout 2 and Bout 3). □, SF; , n-6 PUFA; ■, n-3 PUFA. Mean values with unlike superscript letters between diet groups were significantly different (P < 0·05). * Mean values were significantly different within groups (n 6) (P < 0.05). Values displayed as means with their standard errors.
Recovery between contraction bouts
The hindlimb O2 consumption in the SF group was increased during recovery periods 1 and 2 compared with its own baseline (Fig. 4(A)) (P < 0·05). In comparison, O2 consumption returned towards original baseline levels in both the n-3 PUFA and n-6 PUFA groups. Notably, within each recovery period, O2 consumption for the n-3 PUFA group was significantly lower than that for the SF group (P < 0·05).
Discussion
In rats fed fish oil, skeletal muscle was resistant to fatigue during continuous muscle twitch contractions and recovered contractile force better between repeat bouts. Importantly, the improved contractile performance in the fish oil-fed animals was achieved with reduced skeletal muscle O2 consumption both during contraction bouts and during recovery. These changes in muscle function were associated with the incorporation of n-3 PUFA into skeletal muscle membrane which reflected the well-documented membrane phospholipid responses to the dietary fish oil in excitable tissue(Reference Charnock28) including heart and skeletal muscle in the marmoset monkey(Reference Charnock, Abeywardena and Poletti6), rat(Reference Ayre and Hulbert8) and human subjects(Reference Storlien, Baur and Kriketos29).
Skeletal muscle membrane fatty acid composition
Large differences in skeletal muscle membrane fatty acid composition were achieved with 8 weeks of dietary manipulation; however, there was little indication in the present study that this was specific to muscle fibre type as previously suggested(Reference Kriketos, Pan and Sutton12, Reference Okano, Matsuzaka and Shimojo30). There was some reduction in the concentration of SFA palmitic acid (16 : 0) of red fibres compared with that of the white fibres, which has been inversely related to insulin sensitivity(Reference Vessby, Tengblad and Lithell31). In addition, when provision of essential fatty acids was low (SF feeding), the concentration of DHA was retained at higher levels in the red muscle compared with that of the white muscle. That is, the high oxidative fibres retained higher DHA concentrations compared with the low oxidative fibres(Reference Kriketos, Pan and Sutton12).
The long-chain n-3 PUFA DHA was the major fatty acid incorporated into skeletal muscle membranes with fish oil feeding, and the changes were comparable to those seen in the heart after similar dietary interventions(Reference Pepe and McLennan5, Reference Charnock, Abeywardena and Poletti6, Reference Abdukeyum, Owen and McLennan16). Although the present study used a DHA-rich tuna fish oil supplement, it is evident that DHA incorporation is always the primary response of excitable tissue to long-chain n-3 PUFA fatty acids in the diet, even when EPA-rich fish oils or purified EPA are consumed(Reference Pepe and McLennan5, Reference Charnock, Abeywardena and Poletti6). A systematic comparison of tissue fatty acid composition of the marmoset monkey demonstrated remarkably similar fatty acid profiles between heart and skeletal muscle(Reference Charnock, Abeywardena and Poletti6), whereas other tissues such as vascular smooth muscle, platelets or kidney do not reflect the same extent of DHA incorporation. The DHA concentration in skeletal muscle of the marmoset monkey is very similar to the red and white skeletal muscle of the rat in the present study and further similarities were seen in both SF and n-6 PUFA groups, where DHA concentrations were lower and arachidonic acid concentrations were higher(Reference Charnock, Abeywardena and Poletti6). The similarity in fatty acids of heart and skeletal muscle merits the consideration that skeletal muscle could be used as an indirect marker of the heart n-3 PUFA composition(Reference Charnock, Abeywardena and Poletti6). It is already established that the fatty acid composition of human skeletal muscle reflects the diet(Reference Andersson, Nalsen and Tengblad32) and further use of the biopsy technique in skeletal muscle could allow for indirect monitoring of changes in the heart of human subjects.
Skeletal muscle function
This is the first study to investigate skeletal muscle function on the basis of the modulated O2 consumption previously demonstrated in the heart(Reference Pepe and McLennan17), and its success is largely dependent on properties of the in vivo autologous hindlimb model(Reference Hoy, Peoples and McLennan25), adapted from canine muscle fatigue experiments(Reference Stainsby and Welch33). Only a few studies have considered the effects of dietary-induced changes in membrane composition on skeletal muscle function(Reference Ayre and Hulbert8, Reference Ayre and Hulbert34, Reference Ayre and Hulbert35). These studies used large muscle bundles in vitro, not reflective of in vivo O2 delivery(Reference Zhang, Bruton and Katz36) or voluntary exercise in vivo and produced contradictory outcomes.
The progressive fatigue of the gastrocnemius–plantaris–soleus muscle bundle in vivo during repetitive twitch contractions was less evident in animals fed the n-3 PUFA diet, and fatigue resistance became even more apparent in repeat contraction bouts. Twitch force recovery between contraction bouts was also greater with n-3 PUFA feeding. This is comparable to the enhanced cardiac contractile function(Reference McLennan, Barnden and Abeywardena14, Reference Charnock, McLennan and McIntosh15, Reference Demaison, Blet and Sergiel37) and improved post-ischaemic cardiac recovery(Reference Abdukeyum, Owen and McLennan16, Reference Pepe and McLennan17) seen in association with fish oil feeding.
The inherently low resting O2 consumption of perfused hindlimb was not affected by diet and concurs with findings from O2 consumption of the K+-arrested, quiescent heart(Reference Pepe and McLennan17). In contrast, O2 consumption of the contracting hindlimb, evaluated during the final twitches of each contraction bout, increased in SF and n-6 PUFA hindlimbs, despite the decline in contractile function, but remained low in the n-3 PUFA group although muscle contraction was declining less. O2 consumption and efficiency were modulated when the muscle was put under conditions of increased O2 demand just as is observed in myocardium under stress such as increased filling pressure or acute myocardial ischaemia and reperfusion(Reference Pepe and McLennan17). This represents a clear increase in efficiency of muscle O2 use, again mimicking the effects observed in the heart(Reference Abdukeyum, Owen and McLennan16) where stress-induced increases in O2 use is moderated by fish oil feeding.
Like fish oil feeding in the present study, endurance training promotes greater recovery from bouts of exercise(Reference Hagberg, Hickson and Eshani38). O2 consumption, CO2 production, minute ventilation and heart rate all show faster adjustment to relative and absolute exercise levels and recovery after exercise. Fish oil supplementation reduces whole body O2 consumption and heart rate throughout prolonged exercise bouts in trained cyclists(Reference Peoples, McLennan and Howe23). The observations in the present study suggest that fish oil, like exercise training, may reduce fatigue and improve recovery after exercise.
The similarities between fish oil influences on skeletal muscle and myocardial composition and function suggest common underlying mechanisms. Identification of mechanisms of n-3 PUFA effects is more advanced for myocardium than for skeletal muscle. A vast array of n-3 PUFA-responsive cell signalling processes has been identified, including changes in eicosanoid and free-radical production(Reference Siddiqui, Harvey and Zaloga2). Irrespective of the intracellular signalling, the n-3 PUFA are available through their prior incorporation into phospholipids of the cell membranes. Altered cellular Ca2+ handling is implicated in many of the effects of dietary fish oil in the heart(Reference McLennan and Abeywardena39), including appearing as a common endpoint or intermediate for effects on myocardial intracellular signalling(Reference Siddiqui, Harvey and Zaloga2). Excessive O2 consumption, cardiac arrhythmias and their modulation by n-3 PUFA have all been associated with conditions of cellular sarcoplasmic reticulum and mitochondrial Ca2+overload(Reference Pepe and McLennan17, Reference Pepe, Tsuchiya and Lakatta40). Although skeletal muscle does not mimic the Ca-induced Ca-release of myocardium, it still makes use of the basic properties of Ca release from the sarcoplasmic reticulum into the cytosol and re-uptake which is implicated in muscle fatigue(Reference Favero41). Therefore, Ca2+ cycling processes appear as one potential mechanism of action of fish oil fatty acids in excitable cell function that may translate to skeletal muscle.
The higher unsaturation index of skeletal muscle from fish oil-fed rats implies an increased risk of cellular oxidative damage, which might translate into poor contractile performance in line with the free-radical theory of fatigue(Reference Ferreira and Reid42). The paradoxical fatigue resistance provoked by fish oil feeding suggests that like ischaemic preconditioning(Reference al Makdessi, Brandle and Ehrt43), and nutritional preconditioning by n-3 PUFA in the heart(Reference Abdukeyum, Owen and McLennan16), or exercise training(Reference Pansarasa, D'Antona and Gualea44), the n-3 PUFA up-regulate the endogenous protective mechanisms in skeletal muscle.
In summary, using the rat in vivo autologous pump-perfused hindlimb, fish oil feeding delayed muscle fatigue and improved contractile recovery in association with changes in membrane phospholipid fatty acid composition. As previously observed in heart, there is evidence for the modulation of skeletal muscle O2 consumption associated with increased membrane DHA levels. It is of primary interest to further investigate the levels of fatigue and the corresponding O2 consumption at the commencement of contraction and establish that these findings are reproducible at all points of isometric fatigue. Additionally, it is of interest to establish if these observations also hold true when the skeletal muscle is subjected to greater levels of contractile fatigue ( < 50 % maximum twitch tension) and longer duration of stimulation, which will also impact on the degree of fatigue. The present findings demonstrate the direct implications for dietary fats on skeletal muscle contractile performance and may have applications in cases of muscle fatigue and dysfunction such as heart failure and muscle dystrophy.
Acknowledgements
This project was supported by Australian Research Council Strategic Partnerships with Industry – Research and Training (ARC-SPIRT); Smart Foods Centre and Clover Corporation. The DHA-rich fish oil was provided by Clover Corporation. Each author contributed equally to the manuscript preparation. We declare that there are no conflicts of interest.
