1The Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham; and 2Cardiovascular and Gastrointestinal Global Discovery Research Department, AstraZeneca Pharmaceuticals, Alderley Park, United Kingdom
Submitted 1 October 2003 ; accepted in final form 21 September 2004
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ABSTRACT |
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acetylcarnitine; oxygen deficit; oxidative phosphorylation; phosphocreatine; sodium acetate
A recent series of studies by our group has demonstrated the existence of metabolic inertia at the onset of contraction (27, 28, 3336). On the basis of evidence from these studies, we believe that the delay in acetyl-CoA provision at the onset of exercise, which we have termed the "acetyl group deficit" (27), is a principal determinant of the oxygen deficit (27, 33, 34). It is our assertion, therefore, that the acetyl group deficit reflects a period at the onset of contraction when acetyl-CoA availability fails to meet the increased demands of the tricarboxylic acid (TCA) cycle, thereby limiting oxidative ATP resynthesis and resulting in an increased contribution by ATP delivery from oxygen-independent routes (27, 33, 34, 36). In our most recent study (27), the acetyl group deficit was clearly characterized by the failure of acetyl-CoA or acetylcarnitine to increase during the first minute of contraction, with strong trends for both acetyl-CoA (P = 0.06) and acetylcarnitine (P = 0.08) to decline during this period. Furthermore, this lag in acetyl group delivery came as a result of, and was mirrored by, a lag in pyruvate dehydrogenase complex activation at the onset of contraction (27).
The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible reaction that commits pyruvate to its oxidative fate inside the mitochondrion. As a consequence of PDC activation at the onset of muscle contraction (4, 6), acetyl-CoA delivery via the PDC reaction is markedly increased, providing a stream of substrate toward the TCA cycle for subsequent mitochondrial ATP resynthesis via the electron transport chain. Alternatively, PDC-derived acetyl groups can be transferred to the cellular carnitine pool, presumably when acetyl-CoA production exceeds its rate of utilization by citrate synthase (3). Buffering acetyl groups in this way has been said to maintain a viable pool of free reduced coenzyme A (CoASH) for PDC flux to continue and creates a readily available reservoir of substrate (in the form of acetylcarnitine) for the TCA cycle to subsequently utilize (20). After PDC activation during contraction, it would appear that acetyl group delivery is no longer limiting toward TCA cycle flux. This point is exemplified by the recovery of acetyl-CoA to its resting concentration and the almost linear increase in acetylcarnitine concentration during contraction after PDC activation (17, 24, 27).
We have demonstrated that pretreatment of canine skeletal muscle with sodium dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase (32), near maximally activated the PDC and acetylated muscular carnitine and free CoASH pools at rest (27, 34, 35, 36). During subsequent submaximal ischemic contraction (blood flow and, hence, oxygen availability held at its resting state), dichloroacetate overcame the acetyl group deficit, reduced ATP resynthesis from oxygen-independent routes, and improved the maintenance of contractile function over the course of contraction compared with control (27, 34, 35, 36).
Because dichloroacetate both activates the PDC and acetylates the free CoASH and carnitine pools at rest, it was not possible to determine from this series of studies whether the reduction in oxygen-independent energy production during contraction was the direct result of acetyl group delivery via the PDC reaction being increased from the immediate onset of contraction and/or was attributable to the stockpile of acetyl groups generated at rest, providing a readily available pool of substrate for the TCA cycle throughout the rest-to-work transition. With this question in mind, the aim of the present study was to further characterize the acetyl group deficit by investigating whether, in contrast to the effect of dichloroacetate, pharmacologically increasing resting acetyl group availability, independently of PDC activation, could overcome metabolic inertia, reduce oxygen-independent ATP production, and positively affect function in contracting canine skeletal muscle. In particular, we wished to investigate the time course of PDC activation, acetyl group accumulation, substrate utilization, and tension development during 5 min of electrically evoked ischemic muscular contraction. This was done in the absence and presence of pretreatment with sodium acetate, a known PDC independent acetylator of muscle carnitine and free CoASH pools (7, 17, 25).
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METHODS |
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After the completion of the surgical procedures, each animal was infused with either 30 ml of saline (CON, n = 6) or 360 mg/kg body mass sodium acetate (acetate, n = 6) in 30 ml of saline over a period of 30 min. The dose of acetate used in the present study has been shown previously to near maximally acetylate carnitine and free CoASH pools in human skeletal muscle at rest (7, 17, 25).
Immediately after CON or acetate treatment, the left gracilis muscle was stimulated to contract, under partial ischemia, via electrical stimulation of the obturator nerve (Grass S88 stimulator; Quincy, Medfield, MA). Square-wave impulses of 0.1-ms duration, 3-Hz frequency, and 6-V submaximal voltage were applied for 5 min, resulting in complete muscle fiber recruitment (35). This stimulation protocol produces a workload of 80% maximal oxygen uptake (
O2 max) within gracilis muscle with normal blood flow intact (35). After the experiment, each animal was killed humanely while still under anesthesia by the infusion of pentobarbitone and saturated potassium chloride.
Muscle sampling and analyses.
Immediately before contraction, a resting muscle biopsy was taken by superficial excision of tissue from the distal end of the left gracilis, using a scalpel blade and forceps. Subsequent muscle samples were excised after 20, 40, 60, 180, and 300 s of continuous contraction. All excised muscle tissue was immediately frozen (<2 s) by submersion in liquid nitrogen. Biochemical and histochemical analyses have shown that the canine gracilis muscle (typically weighing 25 g in a 1-yr-old beagle dog) has a fiber type composition of
40% type I and
60% type IIa fibers (22). The canine gracilis muscle is comparable in fiber type composition to human skeletal muscle, and the model used here allows multiple biopsy sampling without a severe detriment to contractile function (27, 28, 3436).
All biopsy samples were divided into two pieces under liquid nitrogen. Subsequently, one portion was freeze dried, dissected free from visible blood and connective tissue, and powdered. After extraction in 0.5 M perchloric acid containing 1 mM EDTA, the supernatant was neutralized with 2.2 M KHCO3 and used for the determination of ATP, PCr, creatine, and lactate (13). The extract was also used for the determination of free carnitine, acetylcarnitine, free CoASH, and acetyl-CoA (2). Freeze-dried muscle powder was also used for the determination of muscle glycogen (13). The remaining portion of frozen wet muscle was used to assess PDC activation (5).
Calculations and statistics.
All data are reported as means ± SE. Comparisons between treatments were carried out using two-way analysis of variance (ANOVA) with repeated measures. When a significant F value was obtained (P < 0.05), a least significant difference post hoc test was used to locate any differences (SPSS Base 8.0). With the exception of lactate, the concentrations of all muscle metabolites were adjusted to the mean total creatine concentration within each individual animal (9). Changes in muscle ATP, PCr, and lactate concentrations were summarized as the magnitude of oxygen-independent ATP production, in accordance with the following formula (31)
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To minimize the number of animals killed for this work, the CON group was shared with another study performed using the same experimental protocol but with dichloroacetate infusion (27). To alleviate any bias, all experiments were performed on consecutive days, and treatments were randomized between CON, acetate, and dichloroacetate infusions (27).
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RESULTS |
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PDCa increased during contraction in both groups, with no significant differences in its activation status existing between treatments at any time point (Fig. 1C). Despite the similarities in activation status, the transformation of PDC to PDCa was faster in CON, increasing significantly from rest after 40 s (CON rest = 0.13 ± 0.05 vs. CON 40 s = 1.10 ± 0.25 mmol acetyl-CoA·min1·kg wet muscle1; P < 0.05) compared with 60 s in the acetate group (acetate rest = 0.18 ± 0.03 vs. acetate 60 s = 1.00 ± 0.27 mmol acetyl-CoA·min1·kg wet muscle1; P < 0.05).
No differences in muscle glycogen concentration existed between the treatment groups at rest or at any time point during contraction (Table 1). Both groups showed a similar profile of degradation during contraction, with the concentration of glycogen falling from rest after 3 min (P < 0.05).
ATP concentrations were maintained during the majority of the period of contraction in both groups (Table 1). However, after 1 min of contraction, the concentration of ATP in the acetate-treated group was significantly better maintained than in the CON group, where it fell from its resting concentration after 5 min of contraction (P < 0.05; Table 1).
Resting PCr concentration was similar between groups (Table 1). After 20 s of contraction, PCr had fallen from its resting concentration in both groups (P < 0.01; Table 1). However, the rate of PCr hydrolysis was markedly reduced in the acetate group over the initial 60 s of contraction, with the concentration of PCr better maintained in the acetate treatment group compared with CON after 20, 40, and 60 s of contraction (P < 0.05; Table 1). No differences existed between groups in PCr concentration after the first minute of contraction (Table 1). Changes in the concentration of PCr mirrored changes in creatine concentration, with no difference in the concentration of the total creatine (sum of PCr and creatine) pool existing within and between groups (Table 1).
No difference in muscle lactate concentration existed between treatment groups at rest. Muscle lactate concentration remained unchanged from rest during the initial 20 s of contraction in the acetate group but more than doubled in concentration over this time in the CON group (P < 0.05; Table 1). However, after 5 min of contraction, muscle lactate concentration was significantly greater in the acetate treatment group compared with CON (P < 0.05; Table 1).
The contribution of oxygen-independent ATP production toward energy production over the entire 5 min of contraction was no different between treatment groups (CON = 122 ± 9 vs. acetate = 148 ± 14 mmol ATP/kg dry muscle). The calculated rate of oxygen-independent ATP production was significantly lower during the first minute of contraction after acetate infusion (CON = 60 ± 2 vs. acetate = 40 ± 8 mmol ATP·min1·kg dry muscle1, P < 0.05; Fig. 2). From 1 to 3 min, no difference existed between groups (Fig. 2), and during the final 2 min of contraction, ATP production via this route was greater after acetate treatment (CON = 13 ± 2 vs. acetate = 32 ± 6 mmol ATP·min1·kg dry muscle1, P < 0.05; Fig. 2).
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DISCUSSION |
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Sodium acetate and acetyl group availability. In agreement with previous studies, acetate increased muscle acetyl-CoA and acetylcarnitine concentrations at rest (P < 0.01) and throughout contraction (P < 0.05) in CON (Fig. 1, A and B; see Refs. 7, 17, 25). Furthermore, and in accordance with our aims, acetate acetylated the carnitine and free CoASH pools to the same extent as pretreatment with dichloroacetate within this animal model (27, 34, 36) and, in contrast to dichloroacetate, achieved this independently of any alteration in PDC activation at rest (Fig. 1C).
Studies that have investigated acetyl group utilization during moderate-to-intense skeletal muscle contraction after sodium acetate pretreatment have reported no reduction in the requirement for oxygen-independent ATP production at any contraction time point (7, 17, 25), implying that elevating acetyl group availability, independently of PDC activation, cannot overcome the period of metabolic inertia and thereby accelerate the onset of mitochondrial ATP production. These previous findings are rather at odds with those reported in the present study; however, this lack of concordance can be accounted for, and on several levels. First, previous studies have failed to examine changes in acetyl group availability at any time point during contraction before PDC activation (7, 17, 25), i.e., to examine the functional role of increasing substrate reserve (in the form of acetylcarnitine and acetyl-CoA) during the period of contraction when they are known to be limiting toward the demands of the TCA cycle. Second, and perhaps more important, previous studies appear to have utilized exercise workloads that are either too low (7, 25) or too intense (17) to optimally assess the functional and metabolic consequences of increasing substrate reserve through acetate infusion. We can say this, as it would appear that metabolic inertia, at least in the form of an acetyl group deficit, only exists over a narrow and predictable range of exercise intensities (between 65 and 90%
O2 max; see Ref. 27), above and below which increasing resting acetyl group availability (in both the absence and presence of PDC activation) will be ineffective at reducing oxygen-independent ATP production and thereby improving contractile function (19, 27). By way of example, at workloads below
65%
O2 max, it appears that PDC flux and/or fat-mediated acetyl group delivery is sufficient to match the energy demands at all contraction time points (7). This is substantiated by the absence of any changes in muscle acetylcarnitine and acetyl-CoA concentrations during 30 s of submaximal exercise (65%
O2 max) after sodium acetate and saline (control) infusion in healthy volunteers, despite PDC activation increasing significantly from rest during this period (7). Similarly, at workloads above
90%
O2 max, where the PDC is activated within as little as 5 s of contraction (1), it appears that acetyl-CoA availability is at no point limiting toward the demands of the TCA cycle, typified by the almost linear accumulation of acetylcarnitine and acetyl-CoA throughout contraction (15, 16). Therefore, on scrutiny of previously published literature, it remains unclear whether increasing the provision of substrate toward the TCA cycle at rest, independently of PDC activation, will reduce the reliance on ATP resynthesis from oxygen-independent routes and improve contractile function. The present study has addressed this issue by using an exercise workload and including contraction time points at which an acetyl group deficit is known to exist.
First minute of contraction. Previous work in this model, where dichloroacetate has been used to increase acetyl group availability, found that oxygen-independent ATP synthesis is markedly reduced during the first 3 min of submaximal ischemic contraction and can be quantitatively accounted for by increased acetyl group utilization after dichloroacetate infusion (27). It would be interesting, therefore, to calculate whether the reduction in oxygen-independent ATP resynthesis seen during the first 60 s of contraction after acetate pretreatment in the present study can be accounted for by the increased availability of acetyl groups.
According to the in vitro-assessed Michaelis-Menten constant (Km) of citrate synthase for acetyl-CoA (2500 µmol/l; Ref. 23), the increased availability of acetyl groups after acetate pretreatment (5.5 mmol/l intracellular water) would, if freely available, markedly promote acetyl-CoA entry into the TCA cycle, via citrate synthase, on the initiation of contraction compared with CON. Indeed, with the assumption that 1 mmol of acetyl groups will produce 12 mmol of ATP equivalents, the sequestering of an additional
1.7 mmol acetyl groups/kg dry muscle by the TCA cycle would be required to account for the difference in oxygen-independent ATP resynthesis (
20 mmol ATP equivalents/kg dry muscle) observed between treatment groups during the first minute of contraction (P < 0.05; Fig. 2). Because the mean concentration of acetylcarnitine appeared to fall by 2.0 mmol/kg dry muscle during the first 60 s of contraction in the acetate group (Fig. 1B), the results appear to indicate that the increased concentration of acetylcarnitine, with a modest contribution from acetyl-CoA (
8.0 µmol/kg dry muscle; Fig. 1A), overcame an acetyl group deficit during this period and thereby reduced the demand for ATP production from oxygen-independent routes (Fig. 2).
The amount of PDC existing in its active form (PDCa) increased substantially in both groups during the first minute of contraction, but no difference in the magnitude of activation existed between treatments at any time point during contraction (Fig. 1C). However, despite these similarities in activation status, the rate of transformation of the PDC to its active moiety was greater in CON, such that PDCa was significantly increased above rest after 40 s of contraction in CON compared with 60 s after acetate pretreatment. Although activation is a prerequisite for an increase in flux through the PDC, differences in the rate of activation between groups during the first minute of contraction do not necessarily equate to differences in flux, which is further controlled by the availability of various reaction substrates and products as well as numerous cofactors (39). The slower transformation of PDC to its active form after acetate is likely to be due to the effect of the high resting acetyl-CoA concentration on the ratio of the two key PDC regulatory enzymes: pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). The antagonist action of these enzymes determine the proportion of PDC existing in its active, dephosphorylated form. A high acetyl-CoA-to-free CoASH ratio, as observed after acetate treatment, is a positive allosteric modulator of PDK activity and would result in a high degree of PDC phosphorylation and inactivation at rest (39). This would have the effect of rendering the PDC more difficult to dephosphorylate and thereby reactivate at the immediate onset of contraction by the intrinsic PDP. Therefore, it is envisaged that resting PDK activity would have been greatly increased after acetate infusion, resulting in the slower transformation of PDC that was observed during the first 60 s of contraction.
Between 1 and 3 min of contraction.
The amount of PDCa increased substantially in both groups during the first minute of contraction. The concentration of acetylcarnitine and acetyl-CoA remained significantly elevated above CON throughout contraction after acetate (Fig. 1, A and B). Despite this increased availability of acetyl groups, the reduction in oxygen-independent ATP regeneration observed during the first minute of contraction in the acetate group was not maintained (Fig. 2), which is in contrast to what we previously have observed after dichloroacetate administration (27, 34). For example, in the study of Roberts et al. (27), which was conducted under experimental conditions identical to the present study, acetylation of carnitine and free CoASH pools at rest, brought about by activation of the PDC with dichloroacetate, reduced oxygen-independent ATP resynthesis by 20 mmol/kg dry muscle between 1 and 3 min of contraction compared with CON. Collectively, these findings imply that an abundance of substrate was available to the TCA cycle after acetate infusion but not utilized. To comprehend why, we need to understand the mechanism by which acetate infusion increases cellular acetyl-CoA and acetylcarnitine concentrations at rest (Fig. 4).
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Between 3 and 5 min of contraction.
During the final 2 min of contraction, no differences in PDCa and glycogen degradation existed between groups, with the availability of acetyl-CoA and acetylcarnitine in CON being only slightly lower than that seen in the acetate group after 5 min of contraction (Fig. 1, A and B). However, despite these similarities, ATP delivery from oxygen-independent routes was 40 mmol/kg dry muscle (or 20 mmol·kg dry muscle1·min1) greater in the acetate group during this period (Fig. 2). This observation could reflect differences in work performed by the muscle during this period (Fig. 3) and/or a reduced contribution of PDC-derived acetyl-CoA toward mitochondrial ATP resynthesis compared with the CON group.
The most striking observation during this period of contraction was the 20% improvement in the maintenance of contractile function observed in the acetate group at 5 min compared with CON. The absence of any net reduction in oxygen-independent ATP resynthesis from CON during the 5 min of contraction after acetate would, in accordance with our previous work (27), be expected to reduce contractile function to the same extent as CON at 5 min. It would therefore appear that the reduction in oxygen-independent ATP resynthesis during the first minute of contraction influences subsequent function or, alternatively, that acetate confers some beneficial effect on muscular function, independently of changes in acetyl group availability, i.e., a significant metabolic alkalosis at rest (Fig. 4; Ref. 28). Indeed, bicarbonate treatment is known to be associated with an enhancement in muscular performance, especially during periods of high-intensity exercise of short duration (14), as has previously been described within this animal model (28).
The present study demonstrated that increasing cellular acetyl group availability at the immediate onset of contraction, independently of any alteration in PDC activation status, overcame inertia in mitochondrial ATP production during the first minute of contraction. However, after this first minute, when the PDC was activated to the same extent in both groups, it appears that PDC-derived acetyl-CoA, i.e., flux through the PDC reaction, rather than increased cellular acetyl group availability per se, was primarily responsible for acetyl group delivery to the TCA cycle and therefore regulated the magnitude of contribution of mitochondrial ATP resynthesis to total energy delivery. Acetate administration was also associated with a reduction in the magnitude of isometric tension loss after 3 min of contraction compared with CON.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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