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Why does muscle tissue have relatively constant AMP + ADP + AMP?


I was going over slides of energy expenditure in muscle cells. It mentions that in muscle tissue, the cell's energy charge ([ATP] / [AMP]) is the principle factor controlling glycolytic activity, and also that glycolysis' primary role here is to provide ATP for contraction.

However, the slides also mentions that "Keep in mind that [ATP] + [ADP] + [AMP] remains relatively constant in the cell over short time frames" with no further explanation. I was under the impression that ATP was actively consumed in muscle tissue during intense activity (which is why lactic acid builds up in muscles: oxidative phosphorylation can't keep up with your rate of energy expenditure).

Thus, my question is Are my slides wrong? During muscle activity, is there a general lack or constancy of ATP?


Adenosine triphosphate (ATP) is often thought of as the energy currency of cells. It is not "used up" per se, but energy is released from the conversion of ATP to ADP (adenosine diphosphate), and yet more can be obtained by removing another phosphate to make AMP (adenosine monophosphate).

Here's a chemical schematic of ATP, you can see the three phosphates to the left.

Your slides state that the amount of (ATP + ADP + AMP) remains constant in the muscle, which makes sense because ATP is not used up, but converted to ADP. So as cells use energy, they do not actually reduce the total concentration of (ATP + ADP + AMP). Energy is required to add the phosphates back onto an ADP or AMP molecule - the process is called oxidative phosphorylation in cellular respiration (although this is not the only way).

I hope I didn't misunderstand your question.


ROLE OF CYCLIC AMP IN DEVELOPING BRAIN

KEDAR N. PRASAD , in Biochemistry of Brain , 1980

Summary

Adenosine 3′,5′-cyclic monophosphate appears to be one of the important factors in the induction of neural tissue as well as in the regulation of several differentiated functions. However, the expression of many of the differentiated functions can be increased by agents which do not change the intracellular level of cyclic AMP, indicating that the regulation of some of the neuronal properties involves more than one mode, one of which is the change in level of cyclic AMP. The expression of morphological and some biochemical differentiation appears to be independently regulated, since one can express in the absence of others. The increase in the level of cyclic AMP binding proteins may be one of the important intracellular mechanisms of maintaining high cyclic AMP level during differentiation, since the protein bound cyclic AMP is resistant to the enzymatic hydrolysis. The increase in the level of binding protein is also associated with the specific changes (increase and decrease) in cyclic AMP-dependent and cyclic AMP-independent phosphorylation activity. Cyclic AMP in some way inhibits the expression of certain genes, whereas it increases the expression of others. We have identified those gene products which are increased, those which are decreased, and those which remain unaltered during cyclic AMP-induced differentiation of neuroblastoma cells in culture. Cyclic AMP in some way must turn off cell division at a precise time during development. Indeed, the synthesis and phosphorylation of H1-histone which are linked with cell proliferation are markedly decreased in cyclic AMP-induced differentiated neuroblastoma cells which have stopped cell division. Nerve growth factor (NGF) also induces certain differentiated functions in developing nervous tissue similar to that produced by cyclic AMP. NGF has shown to increase the cyclic AMP level in certain systems. However, the relationship between cyclic AMP and NGF remains to be defined. The exact mechanisms of cyclic AMP-effect during differentiation are still unknown. Cyclic GMP does not appear to be involved in the mechanism of neural induction or in further differentiation. One fundamental question remains obscure. After neural induction what factor(s) initiate the separation of glial and nerve cells, and various types of nerve cell?


Why does muscle tissue have relatively constant AMP + ADP + AMP? - Biology

Chapter 23 Practice Questions

As the time increases since ingestion of the last meal, hormonal changes occur as one leaves the fed state and enters the fasting state. All the following changes and effects of adipose tissue are true EXCEPT

  1. Insulin increases and activates lipolysis
  2. Glucagon increases and increases lipolysis
  3. With long term fasting or exercise, epinephrine increases and activates lipolysis
  4. With long term fasting or exercise, cortisol increases and activates lipolysis

All of the following statements are true about free fatty acids released from adipose tissue EXCEPT

  1. Fatty acids are hydrophobic
  2. Fatty acids are not soluble in blood, the cytosol, or any other water solution
  3. Fatty acids are transported in the blood by albumin
  4. Fatty acids bind to a hydrophilic binding pocket of albumin
  5. Fatty acids are transported in the cytosol bound to proteins

The major pathway used to oxidize free fatty acids in humans is called

  1. The ketone body catabolic pathway
  2. The phosphatidic acid catabolic pathway
  3. Lipolysis
  4. The carnitine:palmitoyl transferase I and II pathway
  5. Beta-oxidation

All of the following about the fatty acyl CoA synthetase reaction are true EXCEPT

  1. Almost all free fatty acids use this reaction to become activated
  2. ATP is a substrate
  3. Free fatty acids are substrates
  4. ADP is a product
  5. Fatty acyl CoA is a product

Free fatty acids from adipose tissue enter the cytosol, become activated, and must enter the mitochondria to be oxidized. All of the following are part of the pathway whereby Acyl CoA in the cytosol becomes a substrate for beta-oxidation EXCEPT

  1. In the cytosol, the acyl group is transferred to carnitine by the enzyme carnitine palmitoyltransferase I
  2. Acylcarnitine enters the mitochondrion using carnitine acylcarnitine translocase
  3. In the mitochondrion, the acyl group is transferred to CoA by the enzyme carnitine palmitoyltransferase II
  4. The free CoA generated in the mitochondria travels back to the cytosol using carnitine acylcarnitine translocase
  5. The product of the carnitine palmitoyltransferase II reaction is oxidized by beta-oxidation

All of the following statements about beta-oxidation are true EXCEPT

  1. The pathway helps to generate energy and acetyl CoA from fatty acyl CoA
  2. The pathway produces NADH and FADH2 as products
  3. The pathway is found in all tissues that contain mitochondria
  4. The rate of the pathway is dependent upon the rate of fatty acyl CoA entering the mitochondria and the amount of NAD + and FAD available
  5. The pathway is reversible

If stearyl CoA (18 carbon fatty acyl group) were oxidized by beta-oxidation, all of the following would result EXCEPT

  1. 8 acetyl CoA
  2. 8 FADH2
  3. 8 NADH
  4. 32 ATP
  5. The uptake of 8 CoAs into Acetyl CoA

In the normal oxidation of an odd chain fatty acid, all of the following would be part of the process EXCEPT

  1. Each odd chain fatty acyl CoA will produce one propionyl CoA
  2. Propionyl CoA enters the mitochondria using the Carnitine: palmitoyltransferase I system
  3. Propionyl CoA is converted to methylmalonyl CoA
  4. Methylmalonyl CoA is converted to succinyl CoA
  5. There is an increase in the number of 4 carbon intermediates in the TCA cycle

There are several factors that regulate the rate of B-oxidation. All of the following make sense EXCEPT

  1. In the fed state, insulin inhibits the release of free fatty acids from adipose tissue and therefor limits substrate for beta-oxidation
  2. In the fed state, insulin will cause dephosphorylation and activation of acetyl CoA carboxylase that produces malonyl CoA, an inhibitor of CPT1
  3. If the ATP/ADP ratio is low, AMP dependent protein kinase will phosphorylate and inactivate acetyl CoA carboxylase
  4. In the fasting state, the low insulin/glucagon ratio will result in the disappearance of malonyl CoA and activation of CPT1
  5. If the ATP/ADP ratio is low, then high concentrations of FADH2 and NADH will inhibit beta-oxidation

Concerning the pathway for ketone body synthesis, all of the following make sense EXCEPT

  1. Three acetyl CoA molecules can become 3-hydroxy-3-methyl glutaryl CoA (HMG CoA)
  2. HMG CoA is used for cholesterol and ketone body synthesis
  3. HMG CoA lyase produces acetoacetate and acetyl CoA
  4. Acetoacetate is oxidized to beta-hydroxybutyrate and acetone
  5. The rate of ketone body production is proportional to the excess acetyl CoA in the liver mitochondria

The production of ketone bodies is normally a way to transport excess acetyl CoA from the liver to other tissues of the body. All of the following help to explain the process EXCEPT

  1. Blood beta-hydroxybutyrate enters the mitochondria non-liver cells where it will be catabolized
  2. Beta-hydroxybutyrate is oxidized to acetoacetate by beta-hydroxybutyrate dehydrogenase, NADH is produced
  3. Acetoacetate is activated using the enzyme acyl CoA synthetase and ATP
  4. Acetoacetyl CoA + CoA yield two acetyl CoAs which usually enter the TCA cycle and produce energy
  5. Acetoacetate is not catabolized by the liver because the liver lacks the enzyme succinyl CoA acetoacetate CoA transferase

Free fatty acid release from adipose tissue and an increase in the concentration of blood free fatty acids are expected in all of the following cases EXCEPT

  1. During a fast because of increased glucagon
  2. During starvation because of increased glucagon, epinephrine and cortisol
  3. During exercise because of increased epinephrine
  4. During stress because of increased glucagon, epinephrine and cortisol
  5. During a regular meal because of increased insulin/glucagon

All of the following would be expected during an extended fast (starvation) EXCEPT

  1. After about a day, blood glucose levels would remain constant at the low end of the normal range
  2. Blood ketone bodies would rise to very high concentrations over the first 20 days
  3. Blood fatty acids would rise during the first 3 days and very little after that time
  4. The use of blood ketone bodies by the brain would spare the use of blood glucose
  5. The catabolism of muscle protein to produce glucose would increase every day

Select the statement that is not true. As a person in a resting state (no exercise) enters a fast after a mixed meal

  1. Most tissues switch from using mostly fatty acids as fuel to using mostly glucose as fuel
  2. The decrease in insulin inhibits the production of malonyl CoA and this activates CPT1 so more fatty acids can enter the mitochondria
  3. The decrease in insulin/glucagon increases blood free fatty acids
  4. The increased utilization of fatty acids causes inhibition of pyruvate dehydrogenase and hexokinase
  5. Exercise would increase both the use of blood fatty acids and blood glucose for energy production

All of the following help to explain the increased production of ketone bodies as a person enters a fast EXCEPT

  1. The insulin/glucagon ratio drops and release of free fatty acid from adipose tissue increases
  2. Decreased insulin results in phosphorylation of acetyl CoA carboxylase and decreased malonyl CoA
  3. Increased fatty acyl CoA enters the liver mitochondria because CPT I is not inhibited
  4. Beta-oxidation produces too much NADH and FADH2 so Beta-oxidation is inhibited
  5. More Acetyl CoA is produced than is needed by the TCA cycle so the excess it used to make ketone bodies

Otto Shape begins a 20-mile run. All of the following are true EXCEPT

  1. The uptake of blood glucose by muscle will increase within a minute and blood glucose will be lowered
  2. Low blood glucose and increased blood epinephrine will both decrease insulin release
  3. Decreased insulin release will increase glucagon release
  4. Blood cortisol, which must be synthesized before it is released, will start to increase within seconds
  5. Cortisol, glucagon and epinephrine will all cause increased release of free fatty acids from adipose tissue

During a 20-mile run there is an increase in the rate at which acyl CoA is taken up by the mitochondria and used by beta-oxidation. Part of the increase can be explained by the increase fatty acid entering the cell and part is explained by all of the following EXCEPT

  1. The more muscles contract, the higher the concentration of ADP and AMP
  2. AMP-dependent protein kinase causes the phosphorylation of acetyl CoA carboxylase
  3. Phosphorylation of acetyl CoA carboxylase inhibits the production of malonyl CoA
  4. The malonyl CoA concentration drops and inhibition of carnitine: palmitoyltransferase II is removed
  5. Much more acyl carnitine is available to use the carnitine-acylcarnitine translocase

Otto shape is in the process of running 20 miles. As he started the race, the uptake and utilization of glucose by muscle cells increased. All of the following help to explain why this happened EXCEPT

  1. The more muscles contract, the higher the concentration of ADP and AMP
  2. An active AMP-dependent protein kinase results in phosphorylation and activation of hexokinase
  3. The more glucose transporters in muscle cell membrane, the more glucose can enter the cell
  4. A lower ATP/ADP ratio activated phosphofructokinase and increased the rate of glycolysis
  5. Even with increased beta-oxidation, there was a decrease in the inhibitors of pyruvate dehydrogenase

Otto Shape is engaged in a 20 mile run. When compared to the resting state, you would expect the exercise to cause all of the following EXCEPT

  1. Increased ADP concentration
  2. Increased ATP synthase activity as a result of increased ADP concentration
  3. Increased pumping of protons into the mitochondria by the electron transport chain
  4. Increased NADH and FADH2 utilization by the electron transport chain
  5. Increased NAD + and FAD available as substrates for beta-oxidation

Your patient suffers from medium chain acyl CoA (MCAD) deficiency. Compared to a normal person, you would expect all of the following EXCEPT

  1. An increase in ketone body synthesis because of inhibition of the TCA cycle
  2. An increase in blood glucose utilization in the fasting state because the cells cannot get enough energy form beta-oxidation
  3. An increase in the rate at which liver glycogen stores are depleted and the time of onset of hypoglycemia
  4. A decrease in gluconeogenesis in the liver because gluconeogenesis depends upon energy supplied by beta-oxidation
  5. Symptoms to appear anytime the patient does not eat regularly

Your patient suffers from type-1 diabetes. If she missed her regular inulin injection, you would expect all of the following EXCEPT

  1. Decreased insulin and increased stress hormones (glucagon, epinephrine, norepinephrine, cortisol, and others)
  2. Increased mobilization of fatty acids from adipose tissue and increased entrance of fatty acids into all cells of the body
  3. Low insulin and high glucagon to both result in the inhibition of acetyl CoA carboxylase and decreased concentrations of malonyl CoA
  4. Low malonyl CoA would result in faster entrance of acyl CoA into the mitochondria, more beta-oxidation, and more acetyl CoA
  5. Increased ketone body synthesis will result in an increase in blood pH

2. Answer: D. Chapter 23, Objective 2: How are free fatty acids transported from adipose tissue to muscle or liver cells?

3. Answer: E. Chapter 23, Objective 3: Name the major pathway used to oxidize fatty acids into acetyl CoA.

4. Answer: D. Chapter 23, Objective 4: What are the reactants and products of the fatty acyl CoA synthetase reaction?

5. Answer: D. Chapter 23, Objective 5: Describe the pathway for transport of fatty acyl CoA in the cytosol to fatty acyl CoA in the mitochondria. Use the terms carnitinepalmitoyltransferase I and II, carnitine, CoA, inner mitochondrial membrane, and carnitine acylcarnitine translocase, CoA in your explanation.

6. Answer: E. Chapter 23, Objective 6: Use the criteria for understanding and describing all pathways to describe B-oxidation: Names: Functions: Substrates: Product: Control Enzymes: Regulation: Compartment(s): Tissues of interest

7. Answer: A. Chapter 23, Objective 7: Given a saturated, straight chain fatty acid, be able to calculate the number of molecules of Acetyl-CoA, FADH2, and NADH produced by B-oxidation. How much ATP would this be equivalent to?

8. Answer: B. Chapter 23, Objective 8: Be able to name the three metabolites and two important cofactors in the conversion of part of an odd chain fatty acid to a TCA cycle intermediate. (Skip the epimerase) reaction.)

9. Answer: E. Chapter 23, Objective 9: What are the major factors that control the synthesis of acetyl-CoA by B-oxidation muscle and/or liver?

10. Answer: D. Chapter 23, Objective 10: Describe the pathway for the synthesis of ketone bodies by naming substrates, the first ketone body made in the pathway, the next two ketone bodies made in the pathway, the intermediate in the pathway that can be used either for ketone body synthesis or cholesterol synthesis, and the enzyme that actually produces the first ketone body as a product. Control? Where does this pathway reside?

11. Answer: C. Chapter 23, Objective 11: Name a few tissues that oxidize ketone bodies. Why not the liver? What happens to blood ketone bodies? Name the intermediates in the pathway from B-Hydroxybutyrate to acetyl CoA. What does the enzyme succinyl CoA:acetoacetate CoA transferase do?

12. Answer: E. Chapter 23, Objective 12: What is the effect of insulin, glucagon, or epinephrine upon lipolysis in adipose tissue?

13. Answer: E. Chapter 23, Objective 13: What happens to the blood levels of fatty acids, glucose, and ketone bodies during an extended fast? Explain how the use of ketone bodies by the brain spares muscle protein.

14. Answer: A. Chapter 23, Objective 14: If a person eats a balanced meal, does not exercise, and then begins a 10 hour fast. What happens to the rate of carbohydrate and fatty acid oxidation in muscle? Assume that the person dose not exercise. What would happen if they began to exercise vigorously after 5 hours?

15. Answer: D. Chapter 23, Objective 15: How can a decrease in the insulin/glucagon ratio explain the increased production of ketone bodies during a fast?

16. Answer: D. Chapter 23, Objective 16: Concerning Otto shape, what hormonal changes occur during the long distance run and how do they affect the release of free fatty acids from adipose tissue?

17. Answer: D. Chapter 23, Objective 17: Concerning Otto shape, during his long distance run the change in the concentration of AMP ensures the increased uptake of fatty acyl CoA into his muscle mitochondria. Explain this using the terms: muscle contraction, ATP, AMP, AMP-dependent protein kinase, acetyl CoA carboxylase, malonyl CoA, inhibition, carnitine:palmitoyltransferase I, and carnitine-acylcarnitine translocase

18. Answer: B. Chapter 23, Objective 18: Concerning Otto shape, during his long distance run the change in the concentration of AMP ensures the increased uptake of glucose into muscle tissue. How does this happen? Use the terms muscle contraction, ATP, AMP, AMP-dependent protein kinase, glucose transporters, and membrane.

19. Answer: C. Chapter 23, Objective 19: Concerning Otto shape, during his long distance run the change in the concentration of ADP causes increased B-oxidation. Explain this using the terms muscle contraction, ADP, ATP synthase, proton gradient, electron transport chain, NADH oxidation, FAD(2H) oxidation, and B-oxidation.

20. Answer: A. Chapter 23, Objective 20: Concerning Lofata Burne: Explain why medium chain acyl CoA (MCAD) deficiency would cause a decrease in ketone body synthesis during a fast. Also, from an energy point of view, explain why MCAD deficiency would increase the utilization of blood glucose by most tissues of the body and why gluconeogenesis in the liver is less than expected.

21. Answer: E. Chapter 23, Objective 21: Concerning Di Abietes, who suffers from Type I diabetes, what is the cause of her disease? What effect does this have upon blood concentrations of glucagon, catecholamines, and cortisol? What effect do these hormones have upon fatty acid mobilization from adipose tissue? What effect does low insulin and high glucagon have upon fatty acyl CoA entrance into liver mitochondria? What is the effect upon B-oxidation? What effect does this have upon ketone body synthesis? What effect does this have upon blood pH?


Application

Factor #1 Application (Molecular Signaling)

If muscle hypertrophy or strength is the primary pursuit of training, conditioning performed around resistance training at a very high intensity can interfere with the adaptive response to resistance training. Training modalities are likely best separated by at least three hours. Preferably, we advise separating them by

24 hours for insurance, but we will describe why in more detail below.

Factor #2 Application (Fiber type, Bioenergetic, and Systemic Considerations)

While we can’t exclusively target specific muscle fiber types in a practical sense, or control our inherited percentages, there are more systemic benefits of low-intensity conditioning practices worthy of mention. Namely, an increased ability of the cardiovascular system to transport and deliver oxygen-rich blood to muscle tissue between sets and sessions of resistance training can benefit the powerlifter. The benefits of a modest degree of cardiovascular fitness for the serious powerlifter can translate to quicker recovery between sets or training sessions, and therefore the opportunity to present an overload through training that could be missed with sub-par levels of cardiovascular fitness. Since this point seems vague, we’d like to qualify it a bit more in relation to the interaction between systemic and local factors.

Many of you are likely familiar with the term “bioenergetics.” This term refers to the process by which organisms use energy to produce work. Regarding muscle physiology, muscle cells (i.e., fibers) utilize three categorical divisions of bioenergetics, based on their relative contribution to work over time: a) immediate (i.e., ATP-PCr), b) glycolytic (discussed above and below), and c) aerobic or oxidative mechanisms. To be brief, these categories are concomitantly active, but to a lesser or greater extent depending on the intensity and duration of exercise. They are ordered above in terms of their relative contribution to producing ATP to perform muscular work over time. To be clear, in terms of time: 1) the ATP-PCr “system” is primarily active during the first 10-20 seconds of muscular work, 2) the glycolytic system is primarily active between 20-120 seconds (depending on the number of glycogen stores, or the size of glycogen granules in a muscle fiber), and 3) the oxidative system, or mitochondrial respiration, is primarily active thereafter. These time frames are simply rounded values and are not absolute, depending on a variety of factors. Regardless, this information is commonly employed to refute the importance of aerobic metabolism (i.e., mitochondrial activity) in relation to powerlifting training, since most powerlifting training occurs in the range of <=

20 seconds. However, in our opinion, it is vital to the experienced trainee, or coach, to understand how PCr is resynthesized to appreciate the importance of the oxidative system in muscle cells as this pertains to powerlifting programming. To be concise, PCr is primarily resynthesized through the provision of ATP via aerobic metabolism. Perhaps the visual below is helpful to drive this point home.

This image demonstrates the relationship between oxidative phosphorylation (ADP + Pi → ATP) and PCr resynthesis (ATP + Cr → PCr). ATP, produced from oxidative phosphorylation, can interact with an enzyme, creatine kinase, which catalyzes the linking of a phosphate group to creatine (Cr above) resulting in PCr. Why does this matter? Well, how quickly and to what extent PCr is resynthesized between sets is at least cursorily related to how effectively oxygen is delivered to muscle fibers between sets and the ability of muscle tissue to transport and utilize this oxygen for the provision of ATP to resynthesize PCr for high-intensity muscle contraction. Clearly, this is important for the production of force during heavy sets of 1-5 reps, since the immediate energy system is the primary system active. That is, the systemic factors related to the transportation of oxygen-enriched blood and local ability of the muscle fibers reduced of PCr during intense muscular work are intricately related and can be rate-limiting in the sense of supplying the ATP important for producing maximum forces in successive sets of powerlifting training. OK, OK, enough of the mumbo-jumbo, how does this pertain to programming? All other things being equal, the powerlifter with greater cardiovascular abilities to transport oxygen, consume oxygen locally (at the muscle fiber level), and utilize this oxygen to resynthesize ATP for PCr resynthesis between sets can theoretically recover faster between sets to produce higher forces within a whole session involving multiple sets. Consequently, this individual can present a more powerful overloading stimulus through training, albeit with a number of assumptions being made. In a sense, this can be considered a greater acute recoverability .

This translates to a greater propensity to provide a more powerful stimulus during overloading sessions of training for the lifter with greater cardiovascular fitness, assuming no decrement in muscle mass, neural, or structural interference through too much concurrent, or endurance training. This means adaptations from a modest amount of endurance training or low-intensity, more aerobic metabolism-based work can translate to quicker recovery between sets. Although the means to this end can look quite different, practically, adaptations like: a) an increased stroke volume (blood pumped per heartbeat), b) greater perfusion of muscle fibers reduced of PCr during intense contraction (increased capillarization of muscle tissue), and c) an increased mitochondrial density in muscle fibers or increased oxidative enzyme content can improve recovery between sets. These are hallmark adaptations of endurance training. Furthermore, glycolytic activity provides upstream signals to increase mitochondrial activity, which are important for this process to occur. Hence, even in context of more voluminous resistance training (e.g., sets of >6 reps), this argument still holds water since glycolytic activity increases as a function of PCr breakdown, and this feeds into increased oxidative metabolism. To summarize, a practical method of increasing the ability to perform muscular work during a resistance training session aiming to increase maximum strength is to improve the ability of the lifter’s cardiovascular system, and local muscle ability, to transport and utilize oxygen for the resynthesis of ATP. Enter, appropriate dosing of conditioning not directly interfering with resistance training adaptations (i.e., sound concurrent training), at least at some points within the macrocycle of training.

Factor #3 Application [Glycogen]

Each type of training (i.e., endurance and resistance) should alternate foci between the primary musculature used during each session, in reference to completed training the day before or the day after. This is to avoid any potential interference in training performance if glycogen stores are reduced to suboptimal levels in muscle used in the successive session. For example, this could mean placing upper body-based conditioning bouts in closer proximity to lower body resistance training or placing lower body-based conditioning bouts in closer proximity to upper body resistance training.

Factor #4 Application [Time between bouts]

Positioning each type of training on, ideally, separate days altogether (

24h) or at least a few hours after one another could help avoid interference. Since residual fatigue from endurance training can potentially reduce the tension developed during the strength portion of training, care should be taken to allow enough time for the perception of fatigue from either bout to dissipate.


A. Glucose Delivery

Skeletal muscle blood flow can increase up to 20-fold from rest to intense, dynamic exercise (7). Since glucose uptake is the product of blood flow and the arteriovenous glucose difference, this increase in blood flow is quantitatively the larger contributor to the exercise-induced increase in muscle glucose uptake since the arteriovenous glucose difference only increases two- to fourfold during exercise (261). In addition to the large increase in bulk flow to contracting skeletal muscle during exercise, there is also recruitment of capillaries which increases the available surface area for glucose delivery and exchange. Ultrasound imaging techniques have been used to characterize exercise-induced increases in microvascular blood volume, an index of muscle capillary recruitment, in rats and humans (56, 133, 281, 307). Although the extraction of glucose across a working muscle in vivo under most conditions is relatively low (2–8%), an increase in tissue glucose uptake has the potential to decrease interstitial glucose concentrations however, the increase in glucose delivery and rapid transfer of glucose from the capillaries to the interstitium through the endothelial pores ensures that interstitial glucose levels are well maintained during exercise of increasing intensity (193).

Studies in the perfused rat hindlimb have demonstrated the importance of increases in perfusion for the contraction-induced increases in muscle glucose uptake (118, 277, 278). The increase in both glucose and insulin delivery, secondary to increased perfusion, contributes to enhanced muscle glucose uptake. Indeed, it has been estimated that this accounts for ∼30% of the total exercise-induced increase in limb glucose uptake in dogs (344). Although plasma insulin levels decline during exercise, the increase in skeletal muscle blood flow may increase, or at least maintain, insulin delivery to contracting skeletal muscle. Muscle contractions and insulin activate muscle glucose transport by different molecular mechanisms (93, 97, 184, 190, 233, 312), and contractions, flow, and insulin have synergistic effects on glucose uptake in perfused, contracting rat muscle (118) and exercising humans (57, 315). In the former at least, the interaction between insulin and contractions appears to be critically dependent on adenosine receptors (305).

The arterial glucose level is the other important determinant of muscle glucose uptake during exercise. Because glucose uptake across an exercising limb follows saturation kinetics with a Km found to be around 5 mM in dog muscle (343) and 10 mM during knee-extension exercise in humans (247), changes in plasma glucose concentration within the physiological range translate almost directly into proportional changes in leg glucose uptake. With prolonged exercise, as the liver becomes depleted of glycogen and gluconeogenesis is unable to fully compensate, liver glucose output is reduced and hypoglycemia can limit muscle glucose uptake (4, 68). In contrast, increasing arterial glucose availability, by ingestion of carbohydrate-containing beverages, results in increased muscle glucose uptake and oxidation during prolonged exercise (3, 149, 196). The increase in glucose diffusion gradient, as well as a potential glucose-induced GLUT4 translocation (86), drives this increase in muscle glucose uptake however, since metabolic clearance rate (MCR = glucose Rd/[glucose]) is also higher during exercise following carbohydrate ingestion, relatively higher plasma insulin (196) and lower plasma nonesterified fatty acids (110) could also contribute to the higher muscle glucose uptake.


B. Glycolysis, Stage 2

We will follow just one of the two molecules of G-3-P generated by the end of Stage 1 of glycolysis, but remember that both are proceeding through Stage 2 of glycolysis.

Reaction 6:This is a redox reaction. G-3-P is oxidized to 1,3, diphosphoglyceric acid (1,3, diPG) and NAD+ is reduced to NADH. The reaction catalyzed by glyceraldehyde-3-phopsphate dehydrogenase is shown below.

In this freely reversible endergonic reaction, a hydrogen molecule (H2) is removed from G-3-P, leaving behind phosphoglyceric acid. This short-lived oxidation intermediate is phosphorylated to make 1,3 diphosphoglyceric acid (1,3diPG). At the same time, the hydrogen molecule is split into a hydride ion (H-) and a proton (H+). The H- ions reduce NAD+ to NADH, leaving the protons behind in solution. Remember that all of this is happening in the active site of the same enzyme!

Even though it catalyzes a reversible reaction, G-3-P dehydrogenase is allosterically regulated. However, in contrast to the regulation of hexokinase, that of G-3-P dehydrogenase is more complicated! The regulator is NAD+ and the mechanism of allosteric regulation of G-3-P dehydrogenase by NAD+ is called negative cooperativity. It turns out that the higher the [NAD+] in the cell, the lower the affinity of the enzyme for more NAD+ and the faster the reaction in the cell! The mechanism is discussed at the link below.

Reaction 7:The reaction shown below is catalyzed by phosphoglycerate kinase. It is freely reversible and exergonic, yielding ATP and 3-phosphoglyceric acid (3PG).

Catalysis of phosphate group transfer between molecules by kinases is called substrate-level phosphorylation, often the phosphorylation of ADP to make ATP. In this coupled reaction the free energy released by hydrolyzing a phosphate from 1,3 diPG is used to make ATP. Remember that this reaction occurs twice per starting glucose. Two ATPs have been synthesized to this point in glycolysis. We call 1,3 diPG a very high-energy phosphate compound.

Reaction 8:This freely reversible endergonic reaction moves the phosphate from the number 3 carbon of 3PG to the number 2 carbon as shown below.

Mutases like phoshoglycerate mutase catalyze transfer of functional groups within a molecule.

Reaction 9:In this reaction (shown below), enolase catalyzes the conversion of 2PG to phosphoenol pyruvate (PEP).

Reaction 10:This reaction results in the formation of pyruvic acid (pyruvate), as shown below. Remember again, two pyruvates are produced per starting glucose molecule.

The enzyme pyruvate kinase couples the biologically irreversible, exergonic hydrolysis of a phosphate from PEP and transfer of the phosphate to ADP in a coupled reaction. The reaction product, PEP, is another very high-energy phosphate compound.

Pyruvate kinase is allosterically regulated by ATP, citric acid, long-chain fatty acids, F1,6 diP, and one of its own substrates, PEP.

In incomplete (aerobic) glycolysis, pyruvate is oxidized in mitochondria during respiration (see the Alternate Fates of Pyruvate above). Fermentationsare called complete glycolysis because pyruvate is reduced to one or another end product. Recall that muscle fatigue results when skeletal muscle uses anaerobic fermentation to get energy during vigorous exercise. When pyruvate is reduced to lactic acid(lactate), lactic acid accumulation causes muscle fatigue. The enzyme Lactate Dehydrogenase (LDH) that catalyzes this reaction is regulated, but not allosterically. Instead different muscle tissues regulate LDH by making different versions of the enzyme! Click the Link below for an explanation.


C. Glucose Metabolism

Once glucose has been transported across the sarcolemma, it is phosphorylated to glucose 6-phosphate (G-6-P) in a reaction catalyzed by HKII. This is the first step in the metabolism of glucose via either the glycolytic and oxidative pathways responsible for energy generation during exercise or conversion to glycogen in the postexercise period. Glucose phosphorylation is another site of regulation and a potential barrier to glucose uptake and utilization. During maximal dynamic exercise, increases in intramuscular glucose concentration suggest hexokinase inhibition and a limitation to glucose phosphorylation and utilization, in association with elevated intramuscular G-6-P concentration, secondary to increased rates of muscle glycogenolysis (156). Similarly, during the early stages of exercise, G-6-P-mediated inhibition of hexokinase appears to limit glucose uptake and utilization (156). As exercise continues, there is an increase in glucose uptake and a decrease in intramuscular glucose concentration as the hexokinase inhibition is relieved by a lower G-6-P concentration (156). Such a mechanism contributes to the explanation for the temporal relationship between the decrease in muscle glycogen and the progressive increase in glucose uptake during moderate intensity exercise (112). That said, the progressive increase in sarcolemmal GLUT4 is also likely to contribute to this increase in glucose uptake during exercise (177). Increasing preexercise muscle glycogen levels, resulting in greater glycogenolysis during subsequent contractions, is associated with reduced rat muscle glucose uptake (117, 249), most likely via effects on glucose utilization mediated by increased G-6-P concentration. However, since GLUT4 translocation during contractions is also affected by muscle glycogen availability (60, 157), the changes in muscle glucose uptake may also be mediated by reduced sarcolemmal glucose transport. It has been more difficult to demonstrate a direct relationship between muscle glycogen and glucose uptake in human skeletal muscle, since alterations in substrate (glucose and NEFA) and hormone levels, secondary to the exercise and dietary regimens used to manipulate muscle glycogen availability, may confound the results obtained (111, 287, 328). However, when substrate and hormone levels are constant, decreased muscle glycogen prior to exercise is associated with increased glucose uptake during exercise (287).

Epinephrine infusion has been shown to reduce muscle glucose uptake during exercise (139, 318). A widely held view is that this is due to inhibition of glucose phosphorylation by elevated G-6-P concentration secondary to greater flux through glycogenolysis (318). However, epinephrine infusion during exercise that commenced with relatively lower muscle glycogen levels resulted in a similar reduction in glucose uptake and no change in muscle G-6-P concentration, suggesting that the effects of epinephrine on muscle glucose uptake may also be partly mediated via effects on sarcolemmal glucose transport (317). It is also possible that epinephrine has a negative effect on the intrinsic activity of GLUT4 (28).

Using radioisotopically labeled glucose analogs and transgenic approaches (GLUT4 and/or HKII overexpression or deletion), Wasserman and colleagues have suggested that glucose phosphorylation is the rate-limiting step for skeletal muscle glucose uptake during exercise (76, 77, 79, 104). The results of some of the transgenic studies are summarized in Figure 3. GLUT4 overexpression, in the absence of HKII overexpression, had little effect on muscle glucose uptake during exercise. Equally, the full effect of HKII overexpression on glucose uptake was dependent on an increase in GLUT4 expression (Figure 3). These studies in mice actually indicate that the ability to phosphorylate the transported glucose is in fact under most circumstances the rate-limiting step in glucose utilization during exercise. However, the extent to which mouse data can be extrapolated to humans is ambiguous because glycogen concentrations in mouse muscle are ∼10-fold lower than in human muscle and glucose uptake is likely more important for energy provision in mouse than in humans in which muscle glycogen is far more abundant. Thus mice, which do not express glycogen synthase and therefore have no muscle glycogen, are able to run as well as WT mice (230), whereas there is no doubt that low muscle glycogen in humans limits performance (23). Furthermore, as mentioned before, mice that do not express GLUT4 have decreased running ability, indicating the importance for glucose as energy source in mice (78). Overall, the role of glucose phosphorylation in regulation of glucose uptake in humans is ambiguous, and glucose phosphorylation is probably only limiting at the onset of exercise or during intense exercise when rapid glycogenolysis causes G-6-P to accumulate and inhibit HKII (156, 175). Thus, to summarize, glucose uptake in muscle during exercise relies on coordinated increases in glucose delivery, transport, and metabolism, and the step that is actually limiting depends on the actual exercise conditions. Of note, the robust increases in both GLUT4 and HKII expression following endurance training (75) are associated with an increase in both insulin-stimulated glucose disposal (75) and glucose uptake during maximal exercise (175).

Figure 3.GLUT4 and hexokinase II (HKII) as determinants of skeletal muscle glucose uptake during exercise. The figure shows that at rest, overexpression of GLUT4 leads to increased glucose uptake independently of HKII expression. During exercise, HKII overexpression leads to increased glucose uptake at normal and increased levels of GLUT4 expression. Furthermore, GLUT4 overexpression does not in itself lead to increased glucose uptake during exercise. On the abscissa, 1 arbitrary unit denotes the average WT level (n = 8–11 per data point). [From Wasserman (316).]


Creatine-Kinase- and Exercise-Related Muscle Damage Implications for Muscle Performance and Recovery

The appearance of creatine kinase (CK) in blood has been generally considered to be an indirect marker of muscle damage, particularly for diagnosis of medical conditions such as myocardial infarction, muscular dystrophy, and cerebral diseases. However, there is controversy in the literature concerning its validity in reflecting muscle damage as a consequence of level and intensity of physical exercise. Nonmodifiable factors, for example, ethnicity, age, and gender, can also affect enzyme tissue activity and subsequent CK serum levels. The extent of effect suggests that acceptable upper limits of normal CK levels may need to be reset to recognise the impact of these factors. There is a need for standardisation of protocols and stronger guidelines which would facilitate greater scientific integrity. The purpose of this paper is to examine current evidence and opinion relating to the release of CK from skeletal muscle in response to physical activity and examine if elevated concentrations are a health concern.

1. Introduction

CK is a compact enzyme of around 82 kDa that is found in both the cytosol and mitochondria of tissues where energy demands are high. In the cytosol, CK is composed of two polypeptide subunits of around 42 kDa, and two types of subunit are found: M (muscle type) and B (brain type). These subunits allow the formation of three tissue-specific isoenzymes: CK-MB (cardiac muscle), CK-MM (skeletal muscle), and CK-BB (brain). Typically, the ratio of subunits varies with muscle type: skeletal muscle: 98% MM and 2% MB and cardiac muscle: 70–80% MM and 20–30% MB, while brain has predominantly BB. In mitochondria there are two specific forms of mitochondrial CK (Mt-CK): a nonsarcomeric type called ubiquitous Mt-CK expressed in various tissues such as brain, smooth muscle, and sperm, and a sarcomeric Mt-CK expressed in cardiac and skeletal muscle [1].

CK also occurs as macroenzymes. Macro-CK type 1 is a complex of CK (most often CK-BB) and immunoglobulin (most often IgG) and is typically greater than 200 kDa in size. Macro-CK type 2 is a polymer of Mt-CK with a molecular mass of greater than 300 kDa [2]. These forms of CK are expressed during disease and/or dysfunction, for example, macro-CK 1 is associated with cardiovascular and autoimmune disease and macro-CK 2 with cancer. CK catalyzes the reversible phosphorylation of creatine to phosphocreatine and of ADP to ATP [3, 4], and as such it is important in regeneration of cellular ATP:

P h o s p h o c r e a t i n e + M g A D P

CK forms the core of an energy network known as the phosphocreatine (PCr) circuit (see Figure 1). In this circuit, the cytosol isoenzymes are closely coupled to glycolysis and produce ATP for muscle activity. The MtCK version is closely coupled to the electron transport chain and can use mitochondrial ATP to regenerate PCr, which readily returns to the cytosol to resupply cytosolic PCr. This shuttle system is critical for the production and maintenance of energy supply and is involved in the metabolic feedback regulation of respiration [5]. It is unsurprising, therefore, that skeletal muscle has high levels of CK that can account for as much as 20% of the soluble sarcoplasmic protein in specific muscles.


Phosphocreatine (PCr) circuit showing the rephosphorylation of creatine (Cr) in mitochondria using ATP derived from oxidative phosphorylation (oxid phos) and subsequent use of mitochondrial PCr by cytosolic creatine kinase (CK) to resupply ATP for muscle activity, adapted from Saks [5].

Until the mid-1990s, determination of serum CK levels was a key tool in the diagnosis of myocardial infarction (MI) in patients presenting with chest pain in emergency departments. Subsequently, the diagnostic role has been replaced, to a certain extent, by the muscle protein troponin. However, raised levels of serum CK are still closely associated with cell damage, muscle cell disruption, or disease. These cellular disturbances can cause CK to leak from cells into blood serum [6]. Measurement of serum CK activity and determination of isoenzyme profiles are still an important indicator of the occurrence of muscle cell necrosis and tissue damage due to disease or trauma [3].

There has been extensive discussion in the literature regarding the significance of raised levels of serum CK following physical exercise in relation to degrees of muscle cell damage or disturbance. While the reason for release of CK into the circulation is clear in cases such as MI, it is less clear why low- to moderate-intensity physical exercise should also result in release of CK into blood serum. It is certainly confusing that resistance training elicits the greatest release of CK and at the same time provides the best route for muscle hypertrophy. Myofibrillar CK-MM is bound to the M-line of the sarcoplasmic reticulum of myofibrils and is also found in the space of the I-band sarcomeres providing support for muscle energy requirements [7]. Thus, the enzyme is normally confined to the muscle cell so the question arises: do raised levels of CK following a period of exercise represent a degree of actual muscle damage and loss of muscle cell integrity, or is there some other molecular explanation that is not permanent cell damage, but a temporal disturbance or disruption to muscle processes? A greater understanding of this issue could have significant implications for exercise strategy and training programme design (performance and recovery). This is true, not only for athletic populations but also for individuals who participate in strenuous exercise as part of their lifestyle. In this paper, we examine current evidence and opinion relating to the release of CK from skeletal muscle tissue into blood serum in response to muscle exercise.

2. Muscle Response to Exercise

Each mature skeletal muscle fibre is a single cell fused from around 100 myoblasts following fusion, myoblasts lose cell division capacity. Skeletal cell numbers are established before birth. These cells are designed to last a lifetime and are not subject to turnover and recycling processes that occur in many other cell types. Growth in muscle mass happens in magnitude only (hypertrophy via growth hormone and testosterone). While hypertrophy is readily reversible (atrophy), loss of muscle cell numbers as a result of damage would be progressively more serious. Muscles are arranged in bundles of various grade and strength that allow for variable muscle force to suit each need relating to maximal or minimum contractions. Motor units consisting of nerve fibre and associated groups of muscle fibres are recruited as required by nerve stimulation. For stronger contractions more motor units are recruited.

Peripheral muscle fatigue is generally viewed, as a result of insufficient energy and availability of key metabolites that enable contracting muscles to meet increased energy demand. Lack of energy and metabolites will result in motor groups that are unable to fulfill the required workload. Thus, the control of peripheral systems is dependent on the prevailing local metabolism in a motor unit, whereas, in the central model of muscle fatigue, neuromuscular mechanisms aim to preserve overall integrity of the system by mechanisms such as motor unit derecruitment. Golgi tendon organs (GTOs) monitor the tension produced by contraction to prevent excess forces by continuous feedback to the central nervous system (CNS). Thus, the CNS is informed by collective feedback mechanisms that include chemical, mechanical, and cognitive cues. The significance of each of these cues will depend on duration and power requirements of muscular activity. While GTO feedback can be overridden by cognitive processes in the CNS, to allow an athlete to increase performance, it is likely that local peripheral systems can prevent the level of excess muscle contraction that could result in failure or damage.

Unaccustomed exercise, particularly eccentric muscle contractions, initiates mechanical muscle damage of varying degrees [8]. Metabolic muscle disturbance is thought to result in release of cellular components through a cascade of events, which begin with depletion of ATP and result in the leakage of extracellular calcium ions into intracellular space, due to both Na-K-ATPase and Ca 2+ -ATPase pump dysfunction. Intracellular proteolytic enzyme activity can increase and promote muscle protein degradation and augmented cell permeability, which allows some cell contents to leak into the circulation [9, 10]. The process of mechanical and metabolic initiated muscle disruption is not entirely understood it is thought to consist of a complex range of events involving increased oxidative stress, inflammatory and immune responses. Loss of cell myofibre proteins into the blood may occurs at several stages along this continuum (see Figure 2). In most cases, isolated mild to moderate damage in otherwise healthy individuals does not appear to cause further problems, and many studies have demonstrated that the body is capable of clearing released muscle components back to baseline levels within 7–9 days [4, 6] (see Figures 3(a)–3(c)).



(a)
(b)
(c)
(a)
(b)
(c) (a) Changes in serum creatine kinase (CK) activity during 90-minute cycling exercise on three consecutive days (Ex1, Ex2, and Ex3), reprinted from Totsuka et al. with permission from American Physiological Society [6]. (b) Creatine kinase (CK) response to eccentric exercise between immobilisation and control group. PRE refers to the baseline period before exercise. Days 1–4 represent the 4-day immobilization and days 5–9 are the recovery period. Reprinted from Sayers and Clarkson [4]. (c) Creatine kinase (CK) activity in women and in men before (pre), immediately after (post), and 15 days after step exercise. +++ Significant difference from preexercise level (

). $$ Significant difference between men and women (

Factors such as temperature extreme, alcohol abuse, or sporadic strenuous exercise, for example, ultra marathons, can result in more severe disturbance and may require medical intervention to prevent permanent renal damage, primarily due the nephrotoxic effects of myoglobin [9]. Some individuals are found to have high levels of serum CK compared to other similar individuals when exposed to the same exercise protocol (including moderate exercise) even when main comparability factors such as gender, age, and training status are accounted for in data analysis. In some cases, this variability may indicate an underlying myosis, but in many other cases the cause is unknown [7]. There appears to be no established link between habitual exercise or acute high-intensity eccentric exercise and raised incidence of kidney dysfunction or muscle disorder in normal healthy individuals, even in the presence of CK levels >20.000 U/L −1 . The contribution of additional factors such as genetic disposition, environmental conditions, or disease may increase the risk of exertional rhabdomyolysis resulting in acute renal failure [13] (see Table 1).

Individuals who regularly participate in high-volume, intense exercise, tend to have significantly raised base levels of CK compared to sedentary and moderately exercising individuals [14]. Raised levels of serum CK were also found in regularly exercising pre-menopausal women compared to similar sedentary individuals [15] this suggests that CK flux into the serum is a natural and normal reaction to regular exercise.

3. Clinical Significance of Raised Serum CK

Base levels of serum CK in general populations are variable 35–175 U/L [16] with ranges from 20 to 16,000 U/L, and this wide range reflects the inconsistent occurrence of subclinical disorders and minor injury, genetic factors, physical activity status, and medication [17].

In examples of rhabdomyolysis (clinically diagnosed muscle damage) CK levels have been found at 10,000–200,000 U/L and as high as

0 6 U/L [18]. Such levels clearly signal strong disturbance or disintegration of striated muscle tissue with concomitant leakage of intracellular muscle constituents into the circulation. In the absence of specific myocardial or brain infarction, physical trauma, or disease, serum CK levels greater than 5,000 U/L are generally considered to indicate serious disturbance to muscle [10]. It has been recommended acceptable upper limits of normal CK levels be increased by 1.5 times the present limits and that muscle biopsy investigations are only necessary when levels are ≥3 times greater than upper limits and in the absence of exercise induced explanations [19]. However, there is no universally agreed or accepted standard. There are many possible reasons for a diagnosis of rhabdomyolysis and accompanying raised CK levels (see Table 1). Most of the conditions in Table 1 can be attributed to disruption of cells/cell membranes, localised hypoxia, and depletion of energy and disruption of electrolyte balance. Raised levels of macro-CK tend to be associated with disease, though they can also be present in apparently healthy individuals [20].

4. CK Marker for Muscle Damage or Performance Capacity

There is extensive debate in the literature concerning the reliability of serum CK level as a marker of muscle damage. Serum CK determinations are normally initial measures of enzyme activity in blood at the time of sampling, and timeline profiles are mostly set and influenced by the requirements of diagnosis of MI and stroke rather than any exercise influence. The mechanism(s) by which CK is cleared from the blood has not been fully elucidated, and it is likely that observed serum CK levels reflect complex interactions associated with energy status and scale of muscle disturbance. Thus, measured serum CK will reflect relative amounts of CK released, degree of enzyme activity of released CK, and the rate of clearance of CK from the serum [15].

In general terms, high serum CK in some ethnic groups may reflect a genetic condition of naturally increased levels of CK muscle tissue activity, which is not related to exercise frequency or muscle disturbance [21]. It has been proposed that higher than normal levels of tissue CK activity may augment the availability of cellular energy and improve myofibril contraction responses [21]. Therefore, high levels of serum CK, in the absence of muscle damage or other pathological conditions, may reflect the level of enzyme tissue activity of the individual.

Serum CK levels alone may not provide a fully accurate reflection of structural damage to muscle cells [22, 23]. Some studies have reported that serum CK levels were affected by hydration status prior to eccentric exercise and varied within subject groups of comparable male volunteers, whilst muscle biopsies revealed similar ultrastructure damage to Z-band muscle fibres. Muscle soreness did not differ between groups [24]. Biopsies are specific only to a small area of investigation and therefore may not represent the universal extent of damage to the muscle groups exercised. Indeed, the biopsy procedure may itself cause damage to muscle fibres. Other additional indirect indices of muscle damage such as magnetic resonance studies and assessment of delayed onset muscle soreness (DOMS) (which include reduced muscle force post exercise, swelling, perception of pain, and reduced range of movement (ROM)) have been utilized in many studies [25, 26] as have other blood chemical markers of inflammation and stress [27, 28]. These additional measures can assist in quantifying and substantiating muscle disturbance parameters.

5. Exercise Type and Muscle Disruption

Low-intensity (LI) exercise (50% of maximal isometric strength) induced less magnitude of muscle damage and decline in muscle performance than maximal eccentric exercise when the same amount of sets and reps were performed (

reps) [29]. Although sets and reps were matched in this study, work volume was not standardised.

Dynamic concentric and eccentric leg extensions were performed by 21 untrained men and women [30]. Higher-intensity (70% 10 RM and 90% 10 RM) exercise, whilst maintaining a constant 150 reps, elicited greater serum CK, glutamic oxaloacetic transaminase, and serum lactate dehydrogenase levels than lower-intensity (35% 10 RM) exercise. Similarly, when duration of work was increased by performing a greater number of reps, whilst maintaining intensity at 70% 10 RM, serum indices of muscle damage were higher. Therefore, as the volume of exercise performed increased metabolic demands, as might be anticipated, indices of muscle damage were augmented. Interestingly, however, when total work performed was equalized by inversely varying intensity and duration, the greatest rise in serum enzyme levels occurred in the highest-intensity exercise with the shortest duration (80% 10 RM, 170 reps) than following the session with longer duration and lower intensity exercise (30% 10 RM, 545 reps). These results suggest that the magnitude of exercise intensity has greater influence on cellular response to exercise-induced muscle damage than the duration. Another research [31] compared equal volumes of high- and low-intensity eccentric leg extensions on untrained subjects. In this study, work volume was equalised using an isokinetic dynamometer. The authors concluded that there was no significant difference in muscle disturbance indicators (except at 24 hrs). However, high-intensity (HI) exercise did elicit larger declines in muscle performance and a slower recovery. This may be due to a greater recruitment of type II muscle fibres in high-intensity eccentric exercise, which have been found to be more susceptible to disruption compared to type I [23, 32]. Serum CK levels were higher with high intensity, but not significantly. Subjective measurement of pain and ROM measurements showed no significant difference between groups. In this study, equal volumes of work result in similar indices of muscle disruption, but with less decrement in muscle performance, and greater recovery with low intensity compared to high intensity. The use of leg extensions in the latter study compared to elbow flexion in Nosaka and Newton’s study [29] may have contributed to the variations in muscle disturbance indices between the two studies. There is evidence to suggest that the degree of muscle damage is greater in elbow flexion compared to knee extension [26]. However, both studies did agree in their findings concerning greater declines in muscle performance after HI compared to LI.

yrs) were randomly assigned to one of four experimental groups using a bench press protocol (

, 110% 1 RM, and a control group

). There was no significant difference in the total volume of exercise among the groups. All subjects showed a significant (

) increase in postexercise CK activity. The highest values occurred at 24, 48, or 72 h however, no significant

difference was found between groups, although there was a large variability between subjects. There was no significant difference in muscle soreness between groups (

). The 110% 1 RM had a significantly higher (

) prostaglandin E2 (PGE2) than the other groups at 24 and 48 h after exercise [33].

This study also concludes that volume of exercise rather than intensity determines the level of muscle damage however, the subjects in the 50, 75, and 90% groups executed eccentric and concentric actions, whereas the 110% group only performed eccentric contractions. This may have influenced the magnitude of muscle damage in the 110% group. Total volume of exercise was determined from a calculation (total

v o l u m e = n u m b e r o f s e t s × n u m b e r o f r e p e t i t i o n s × l o a d

(kg)) therefore the calculated volume may not have been determined as accurately as the isokinetic dynamometer protocol.

The higher levels of PGE2 in the 110% 1 RM group suggest a greater magnitude of inflammation at 24 and 48 h compared to the other groups. Muscle force measurement may have further evidenced any variations in strength deficits caused by variation of 1 RM%.

The variances observed in studies [29–31, 33] may be due to disparities in study methods, and the large variation in CK response within and between studies makes a definitive conclusion on the contribution of intensity and volume of exercise on cell changes difficult. Considering the significant increase in CK levels which have been found as a result of high-intensity exercise compared to lower intensity [29, 30], the decrements in performance experienced [29, 31], and higher levels of PGE2 reported [33] even when exercise volume is standardised suggests that higher-intensity exercise will cause the greater disruption of cell membranes however, with adequate recovery, it may also elicit the greatest adaptations to exercise in the shortest time.

Seven continuous days of the same isokinetic maximal elbow flexion protocol (ECC2 to ECC7) did not increase indices of muscle disturbance compared to a control group who performed only one session of the exercise protocol (ECC1) [27]. Plasma CK levels increased significantly (

) in both groups, peaking 4 days after the initial bout of exercise. There was a decline in levels over the course of the next 6 days, and both groups had insignificant CK plasma levels at day 7 there was no significant difference between groups at any time. This was attributed to increased resistance to muscle stress or to that no further muscle disruption had occurred [27]. Total work was reduced in the ECC2 to ECC7 group at each of the six further exercise sessions compared to the first day of exercise however, they were considered to be of the maximal intensity possible, even if at a lower absolute magnitude.

Despite theories of muscle protection and reduced disruption from further consecutive eccentric disruption afforded by the initial exercise bout in this study, the loss of muscle force which resulted in reduced work load presumably would have influenced the results. It is interesting to consider whether the initial loss of CK contributed to the loss of strength over the 6-day period or whether the loss was associated with disruption to type II fibres.

A number of studies have used very high intensity or volume of exercise, or both, to ensure muscle disruption is elicited [34, 35]. Lower-intensity submaximal muscle voluntary contractions (60% MVC) have been shown to be linearly related to CK levels, muscle oedema, and perception of pain compared to higher intensity (80% MVC) [36]. Evans et al. [36] suggest that greater magnitudes of muscle disturbance may alter time course and correlation between muscle fibre damage, pain, and CK release and may in part account for reports by some studies that CK is not a reliable marker of muscle disruption. It has been proposed that, in fact, moderate levels of force may produce superior measurement parameters [35].

6. Gender Influences on Muscle Damage

Gender difference in muscle disturbance and repair processes has frequently been reported in the literature. Studies on female animals have demonstrated lower baseline levels of CK and an attenuated CK response to exercise [37, 38]. However, females presented with a higher CK peak and a greater relative increase in serum CK levels after 50 maximal eccentric contractions of the arm flexor muscles, despite significantly lower baseline levels compared to males [39]. Thirty minutes of stepping exercise resulted in a CK serum increase in 15 women from a baseline of

U/L at day 3. There was no significant increase in CK serum levels in the 18 men who performed the same protocol (see Figure 3(c)), however, the authors suggest this may in part be due to greater adaptation to this type of exercise in the males [12].

Rinard et al. (2000) suggest that many of the findings that indicate women have an attenuated response to muscle damaging exercise are due to poor study design and may apply more specifically to aerobic exercise and that there is little or no difference between males and females in their response to eccentric-exercise-induced damage [40]. This view is supported in a review by Clarkson and Hubal (2002) who conclude that any differences between genders are small and indicate that females may be more inclined to muscle disruption than males [41].

In postmenopausal women not taking hormone replacement treatment (HRT) [42] and amenorrheic women [15], raised levels of CK in response to exercise-induced muscle disruption were found, when compared with women on HRT and premenopausal women. This effect was attributed to lower oestrogen levels. Oestrogen may be important in protecting cell membranes from damage [11] and reduced infiltration by leucocytes may lessen their damage causing function in the repair process. Conversely, this may also delay the healing process [43]. Leucocytes may have a role in the activation of satellite cells [11] which proliferate and differentiate forming new muscle fibres [44]. Whether oestrogen can promote reduced CK efflux via reduced membrane permeability or whether actual muscle damage is reduced is not clear [43]. Progesterone has been suggested to interact with oestrogen and may antagonise the oestrogen disruption limiting properties [44].

A study by Arnett et al. (2000) examined CK response to unaccustomed eccentric hamstring exercise in premenarcheal (P) and menarcheal girls (M) and postmenopausal (PM) women. Preexercise levels of CK were significantly greater in PM than in P girls or M women, and CK-MB was greater in M than in both P girls and PM women. However, after exercise M women had significantly higher levels of CK and CK-MB than both P girls and PM women at 24, 48, 72, and 96 hours after exercise. This study concluded that oestrogen levels had no significant effect on CK levels after strenuous eccentric exercise [45]. However, knee ROM in subjects was not assessed. Variations in ROM have been suggested as affecting the mechanical strain on the muscle during eccentric exertion [25]. This activity alters the force applied to sarcomeres and modifies the magnitude of disturbance [46]. Work volume in each group was not measured therefore, variations between groups may have occurred, affecting associated muscle disruption, and high baseline CK levels in PM may be related to age variations in energetics.

7. Age-Related Muscle Disruption

Studies of serum CK response to exercise in aging human skeletal muscle have produced variable results. A review by Fell and Williams (2008) on the effect of aging on skeletal muscle in athletes suggests that aging can lead to greater exercise-induced damage and a slower repair and adaptation response [47]. Muscle mass and function gradually decline with age, and cell apoptosis may have a role in age-related sarcopenia [48]. Lower levels of plasma CK in older female subjects have been attributed to a decline in circulating neutrophils with age which may, in part, be due to reduced oestradiol levels and endogenous antioxidant status [45]. Circulating neutrophils produce oxidants such as superoxide free radicals, which increase cell damage and leakage. Therefore, an increased serum CK could be related to optimal functioning of the cell, which may decline with age, and is not simply a marker of less damage. Free radial production appears to moderate signalling for adaptation of skeletal muscle in response to exercise [49], and this response may be attenuated in older muscle, rendering it less adaptive to exercise stress.

Studies on humans have produced conflicting results in relation to aging muscle response to exercise. Some show evidence of more muscle ultrastructure damage in older subjects (

yrs) compared to young (26±1 yrs) [50] and others in relation to less damage in older (

yrs) subjects compared to younger (

yrs) [45]. Lavender and Nosaka (2008) reported no significant changes in indirect measures of muscle damage after unaccustomed eccentric elbow flexion by males (19–25 yrs and 41–57 yrs) [25]. Individual ROM at the elbow was not significantly different between subjects however, during the exercise the investigator assisted subjects in keeping the velocity of the movement constant. This may have affected the magnitude of muscle damage.

Subjects in this study were described as habitually active. Regular physical activity has been shown to slow the process of sarcopenia and may reverse age-related muscle apoptosis [51]. Exercise may also attenuate and protect against exercise muscle disruption and subsequent damage. Therefore, the level of past and present physical activity may significantly affect muscle damage throughout the ageing process. It would be of interest to explore the effects of habitual training in different age groups and its effect on CK serum levels.

Exposure to exercise stress initiates adaptation in gene expression, cellular protective mechanisms, and remodelling, which help protect muscle during subsequent bouts of exercise [49]. The ability of aged muscle to adapt to environmental stress appears to be impaired, as are repair mechanisms, and heat shock protein (HSP) production is reduced in response to physiological stress in animals [49, 52].

Exercise disturbs muscle homeostasis by depleting glycogen, lowering pH, increasing hyperthermia, and increasing ROS (reactive oxygen species) production as a by-product of energy metabolism. These perturbations (or a combination of them) will initiate a stress response which instigates the release of HSPs such as HSF1 and its cochaperones, for example, HSP70 or HSP90 [53].

In particular, higher levels of ROS after exercise can increase the oxidation of thiol (sulphydryl) groups on proteins, leading to increased protein damage, and may trigger release of HSF1 [54]. Once exercise stress has subsided, cochaperone HSPs bind to HSF1 and deactivate it [55]. The instigation of an HSP response is dependent on a number of factors including the type and intensity of exercise, muscles involved, and the age and training status of the individual. The aging process appears to change ATP pathways, alter muscle fibre type ratios, and reduce HSPs response, which are thought to offer some degree of protection against further exercise-induced muscle damage.

8. CK and the AMPK Energy Sensor

AMPK (AMP-activated protein kinase) is an energy sensing enzyme that is widely dispersed in nature from single-cell organisms to humans, is central to the management of energy supply, and operates both locally and at whole organism (see Figure 4). At times of rest/inactivity it is inactive, and metabolic processes focused on synthesis, storage, and accumulation proceed unhindered. When activities occur that deplete ATP levels, such as physical exercise, glucose depletion, or hypoxia, AMPK is activated. When activated, it in turn stimulates a range of physiological and biochemical processes and pathways that increase ATP production and at the same time switch off pathways that involve ATP consumption. Recent work has shown a strong correlation between a sedentary lifestyle, inactive AMPK, and morbidity diseases such as metabolic syndrome, type 2 diabetes, and dementia [56]. The benefits of exercise in providing protection from such morbidity diseases are now firmly linked to activation of AMPK and associated biochemical and physiological processes that are stimulated. The primary activity of AMPK is to phosphorylate proteins especially enzymes and by this action regulate the activity of key enzymes that operate important reactions and pathways.


Potential roles of adenylate kinase (AK) and AMP-activated protein kinase (AMPK) in gross control of creatine kinase (CK) activity by promoting expulsion of CK from the cytosol to limit CK utilisation of ATP for PCr resynthesis adapted from Saks [5].

The role of CK in energy management is maintenance of PCr levels to provide an immediate energy supply in the first few seconds of physical activity. It is likely that AMPK has a role in controlling CK activity, and some work has demonstrated that AMPK may regulate CK and is sensitive to the Cr : PCr ratio and that increased creatine levels stimulate AMPK activity [57]. Given the widespread action of AMPK (during exercise) to switch off nonessential ATP consumption, it is likely that AMPK would act to limit the use of ATP by CK to produce PCr and re-establish the PCr pool [58]. During intense exercise there is no PCr resynthesis and the reaction is likely blocked by more than one mechanism however, although there is no need for PCr resynthesis, there is a need to maintain the ratio and AMPK could be part of the overall process.

It is clear that such a system would not act in isolation but as part of a sophisticated process involving other regulatory functions in the muscle, and only when the full integrated system is understood will it be possible to explain the many anomalies associated with muscle action. For example eccentrically biased exercise (e.g., downhill running) will elicit greater postexercise levels of serum CK than equivalent concentrically biased exercise (e.g., uphill running) though the former is less energy metabolism demanding than the latter [41]. This highlights the integrated complexity of metabolism and mechanical damage as eccentric-biased exercise is associated with increased indices of muscle damage (i.e., DOMS) which is mainly a result of micro-damage within the myocyte [59, 60]. In addition, eccentric-biased contractions may be more mechanochemically efficient based on changes in the actin-myosin sliding length in that changes in sliding length produce different levels of tension and consequent different degrees of muscle damage [61]. It may be that as eccentric and concentric contractions have different demands and consequences on the metabolic and mechanical components of muscle action, there are alternate mechanisms of control via AMPK that produce different effects of CK levels. This would allow maximum flexibility for a wide range of exercise stressors to enable survival of the species in prehistoric environments where survival depended upon adaptable and flexible muscle action.

ATP levels never deplete to critical levels this is because the sensitivity of ATP is set very high to guarantee that they never deplete, so a slight reduction in high ATP level triggers an early protective reaction. It is postulated here that in some circumstances, either directly or indirectly, the controlling activity of AMPK could trigger a process that culminates in the elimination of CK from the cell as part of a mechanism to regulate metabolic and/or mechanical disruption of muscle cells to prevent muscle failure caused by accumulative damage with consequent increase in serum CK levels (see Figure 4). This might be a component function in the overall action of fatigue to limit muscle activity or it could be a system that evolved prior to or in parallel with fatigue mechanisms.

The AMPK mechanism of control involves phosphorylation of CK, and it may be that phosphorylation provides a signal to facilitate removal of CK from the cytosol (see Figure 4). Such a mechanism would explain the appearance of serum CK following physical exercise as opposed to structural damage arising from muscle trauma. Following muscle damaging exercise, CK levels continue to rise in the blood for hours or days (see Figures 3(a)–3(c)) despite significant metabolic disruptions having ceased. The capacity for compromised muscle tissue to generate force is impaired [27, 31, 62] therefore, measures are required to protect and facilitate the repair of muscle tissue. In addition, other processes which disrupt the cell membrane, for example, inflammation, continue [63], allowing CK to exit the cell over time. This extended loss of CK may be associated with protective mechanisms, and a prolonged involvement of AMPK, allowing repair and restoration of muscle function.

9. Influence of Genetic Characteristics

Exercise-induced muscle disruption is known to produce insulin-like growth factor II (IGF II) in response to cell damage and is thought to stimulate satellite cells and hypertrophy. An association has been found between a polymorphism in the sarcomeric protein myosin light chain kinase and changes in blood CK, Mb, and isometric strength, in individuals with specific genetic variations in alleles of IGF II who experienced increased muscle disruption as a result of maximal isotonic eccentric contractions [64]. This suggests that these genome variations may lead to alterations in calcium handling and force effects during exercise, thereby influencing muscle disruption. This could explain the susceptibility of some individuals, who are otherwise healthy, to muscle disruption and exertional rhabdomyolysis [64] and the large intersubject variation in levels of serum CK found in many studies.

Heled et al. (2007) explored the possibility of a genetic association between CK MM, angiotensin-converting enzyme (ACE) genotypes, and CK response to exercise [7].

A genetic association was found between a specific CK-MM genotype of the Ncol polymorphism with an augmented response to exercise.

Yamin et al. (2007) found an association between type of ACE genotype and CK levels. ACE genotypes may be involved in the excitation coupling process and influence the risk for developing rhabdomyolysis and, conversely, protection against exercise-induced muscle injury. However, this effect may be more noticeable in previously sedentary individuals performing intense exercise [65]. Other studies featuring physically active subjects did not find a comparable association [7].

Intensive exercise initiates an immune response resulting in acute and delayed leukocytosis, featuring neutrophils predominantly. It develops approximately 30 minutes after acute exercise, and leukocytosis peaks several hours after exercise before returning to baseline levels 24 hr after exercise [66]. This delayed proinflammatory response may in part be related to the serum CK response observed after exercise-induced muscle damage, due to leucocytes infiltrating and destabilising the cell membrane during the process of repair.

Serum CK followed a biphasic pattern increasing until 23 hr after exercise declining weakly at 47 h before increasing again and peaking 95 h after exercise. This biphasic response has been noted in other studies [23, 35] and may be related to the time line of inflammation.

10. Exercise Modality

Exercise modality can affect the appearance of CK in blood serum. Eccentric resistance training CK serum levels can peak between 72 hrs [31, 45] and 96 hrs [67] to 120 hrs [4] (see Figure 3(b)). Training status may affect this time response. Full body eccentric resistance training in resistance trained (RT) and untrained (UT) men elicited a significant (UT

) increase in CK serum levels at 24 hrs. This signified the peak response in the RT group, whilst levels in the UT group continued to rise and peaked at 72 hrs [68]. However, three sets of 50 maximal eccentric leg flexion contractions in untrained men resulted in a significant (

) increase in CK serum levels at 24 hrs levels decreased over the next 2 days followed by a nonsignificant (

) increase at 96 hr [23], and 10 sets of 10 reps of 70% body mass barbell squats incorporating eccentric and concentric contractions in non-resistance-trained males and females resulted in a peak serum CK response at 24 hr after exercise. A series of plyometric jumps performed over 2–5 minutes by untrained men produced a peak CK serum response at 48 hrs [69], and 90 minutes of endurance cycle ergometer exercise at a set absolute workload (1.5 kilo ponds at 60 revolutions per minute) performed by untrained men three days consecutively caused a significant (

) increase in serum CK levels 3 hours after the first exercise session and peak CK serum levels occurred immediately after the third day of exercise, 72 hrs from the initiation of exercise [6] (see Figure 3(a)). Stepping exercise resulted in a CK serum increase in women at day 3, whereas, there was no significant increase in CK serum levels in men performing the same protocol (see Figure 3(c)).

Pantoja et al. [70] analysed the muscle disruption effect of dynamic resistance training performed on land or in water. The duration of the ten-rep max for elbow flexion for each subject was recorded with a chronometer in order to standardise exercise in both land and water environments and induce the same energy-generating metabolic pathways. Subjects executed as many maximal effort contractions as possible for each set performing three sets in both environments with two-minute rest between sets each environment session (land or water) was separated by four weeks.

A significant increase in serum CK was observed at 48 hours after exercise on land, and no significant change in baseline serum CK levels occurred in water. No further samples were taken after this time. The main mechanism hypothesised to have attenuated muscle damage in water was reduced eccentric contractions [70].

There are difficulties in comparing exercise intensity and work volume in land and water [71, 72]. Standardisation of exercise between water and land is challenging due to the differing conditions in water compared to air (resistance, temperature, and hydrostatic pressure).

The significance of exercise modality on CK serum response appears to be related to the magnitude of eccentric contractions involved in the activity and the subsequent extent of muscle disruption. Greater muscle cell disturbance delays the appearance of a CK serum peak compared to less disruption. This may be linked to the time course of inflammation however, evidence in the literature supporting this theory remains unclear.

11. Conclusion

The molecular mechanisms that result in CK release from muscle after mild exercise are unclear. More clarification could provide important information for athletes concerned about muscle hypertrophy, performance, and the importance of rest periods between periods of exercise. Future studies should include an exploration of ethnic variations in CK response to exercise. In the absence of any mechanical muscle damage, it remains a question as to whether raised CK after exercise does represent a degree of actual muscle damage or some form of disruption in energy control processes or some other molecular reaction mechanism.

Since muscle tissue cannot ignore brain centred nerve stimulations causing increase in both the number of motor units recruited and the frequency of motor unit stimulation, as well as creation of longer tetanic contractions, it would seem logical that muscle would have some mechanism of moderation to delay the final sanction of fatigue for as long as possible. It is considered here that this could be a membrane event in which a proportion of cytoplasmic enzymes/proteins such as CK exit the muscle cell to place a temporary energy restriction and allow subsequent relaxation and regeneration.

A key regulator in this event would be the energy sensor enzyme AMPK, which can phosphorylate CK and is sensitive to Cr/PCr ratios. At the start of physical exercise the initial supply of ATP for muscle activity is provided via the Cr-PCr shuttle by the readily reversible CK catalysed conversion of PCr + ADP to Cr + ATP until PCr is depleted. As physical activity continues and ATP is increasingly produced by oxidative phosphorylation, there is potential for the rapid rise in ATP levels to be blunted if both MtCK and cytosol CK use the ATP to regenerate PCr. Since AMPK has an overall role, during physical exercise, to limit ATP consumption by nonessential systems, it is likely that this extends to CK. Although PCr resynthesis is greatly diminished during high-intensity exercise, AMPK may still be required to maintain the ratio. It is speculated here that the control involves expulsion of CK from the cytosol (see Figure 3). If this is the case, then increased serum CK levels arising from normal physical exercise may be a consequence of normal metabolic activity rather than representative of physical damage to muscle. Further the wide ranges of serum CK found in the population could reflect different levels of sensitivity of AMPK and/or levels of AMPK, resulting in varying levels of control and hence varied expulsion of CK from the cytosol. Such a system would not act in isolation but as part of a sophisticated process involving other regulatory functions in the muscle, and only when the full integrated system is understood will it be possible to explain the many anomalies associated with muscle action.

It is suggested here that the appearance of CK in serum following low- to moderate-intensity exercise represents a disturbance to muscle energy processes and is not representative of the type of muscle cell damage observed following MI, stroke, or other physical/structural damage. Unfortunately, it has not been possible from the available literature to extract more definitive evidence for this suggestion. The considerable variability across many studies makes interpretation more difficult, and it is clear that the lack of agreed guideline procedures and defined parameters for the conduct and evaluation of exercise-based experimental work in this area is a major barrier to the greater understanding of the influence of exercise on muscle and human health in general. The establishment of an international committee on exercise-based experimental and laboratory protocols may be beneficial. Such a committee could provide leadership, clarity, and standardisation that would enable researchers to effectively answer related experimental questions.

Conflict of Interests

The authors have no conflicts of interests that are directly relevant to the content of this paper.

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Copyright

Copyright © 2012 Marianne F. Baird et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Abstract

Endurance training lowers heart rate and blood pressure responses to exercise, but the mechanisms and consequences remain unclear. To determine the role of skeletal muscle for the cardioventilatory response to exercise, 8 healthy young men were studied before and after 5 weeks of 1-legged knee-extensor training and 2 weeks of deconditioning of the other leg (leg cast). Hemodynamics and muscle interstitial nucleotides were determined during exercise with the (1) deconditioned leg, (2) trained leg, and (3) trained leg with atrial pacing to the heart rate obtained with the deconditioned leg. Heart rate was ≈15 bpm lower during exercise with the trained leg (P<0.05), but stroke volume was higher (P<0.05) and cardiac output was similar. Arterial and central venous pressures, rate-pressure product, and ventilation were lower during exercise with the trained leg (P<0.05), whereas pulmonary capillary wedge pressure was similar. When heart rate was controlled by atrial pacing, stroke volume decreased (P<0.05), but cardiac output, peripheral blood flow, arterial pressures, and pulmonary capillary wedge pressure remained unchanged. Circulating [norepinephrine], [lactate] and [K + ] were lower and interstitial [ATP] and pH were higher in the trained leg (P<0.05). The lower cardioventilatory response to exercise with the trained leg is partly coupled to a reduced signaling from skeletal muscle likely mediated by K + , lactate, or pH, whereas the lower cardiac afterload increases stroke volume. These results demonstrate that skeletal muscle training reduces the cardioventilatory response to exercise without compromising O2 delivery, and it can therefore be used to reduce the load on the heart during physical activity.

Introduction

A difference in training status of the exercising skeletal muscle, such as obtained by 1-legged training or immobilization, leads to a markedly lower heart rate (HR) and blood pressure response when 1-legged exercise at the same workload is performed with the best trained leg. 1,2 These observations indicate that training-induced changes in HR and blood pressure responses to exercise are sensitive to other factors than changes in the central circulation (ie, heart size 3,4 and left ventricular function 5,6 ) and that factors within the skeletal muscle 1,7 play a role in the altered cardioventilatory response to exercise with exercise training. However, the regulatory mechanisms and consequences for the training-induced changes in central and peripheral hemodynamics remain unclear.

During exercise, sympathetic nervous activity is increased 8 by influence from a central feed forward mechanism (central command) and contracting skeletal muscle (the exercise pressor reflex), 9 resulting in an intensity-dependent increase in HR, ventilation, blood pressure, and vasoconstriction in the inactive tissues. 9,10 Afferent fibers located within the skeletal muscles respond to mechanical distortion (group III) and changes in the chemical environment (group IV), 11 and are thought to contribute to the cardioventilatory response to exercise. 12 Physical training attenuates the increase in sympathetic nerve activity during exercise 13 and leads to local adaptations, such as an elevated number of capillaries and a higher mitochondrial capacity affecting both aerobic and anaerobic metabolism. 14 Interstitial potassium (K + ), 15,16 pH, lactate, and adenosine have been suggested to contribute to the exercise pressor reflex by stimulating and sensitizing group IV fiber afferents in skeletal muscle, 11 although their individual contributions remain controversial. 11,17,18 Recently, ATP has been suggested to stimulate group IV afferents, 19 and a role for ATP is supported by the tight coupling between interstitial ATP concentrations and exercise intensity 20 and the relation between interstitial ATP and norepinephrine (NE) concentrations during exercise. 21,22

This study investigated the role of the training status of skeletal muscles on cardiovascular responses to exercise. A secondary aim was to evaluate whether muscle interstitial ATP affects afferent feedback from the contracting muscle. We measured hemodynamics and muscle interstitial nucleotides and adenosine concentrations at rest and during exercise with a control leg, a trained leg, and a deconditioned leg. A large difference in muscle training status within the same individual was obtained by training 1 leg for 5 weeks, while the contralateral leg was immobilized for 2 weeks before the final experimental day. The importance of training-induced difference in HR response to exercise was evaluated by increasing HR during exercise with the trained muscle to the same HR as established during exercise with deconditioned leg. Our hypothesis was that the training status of the contracting muscles influences cardioventilatory response to exercise at a given workload by lowering the relative exercise intensity of skeletal muscle and sympathetic activation. Furthermore, our hypothesis was that exercise training alters skeletal muscle interstital ATP signaling, and thereby contributes to attenuated cardioventilatory response to exercise.

Methods

Eight recreationally active male subjects with a mean (±SD) age of 24±4 years, body weight of 77±11 kg, height of 184±7 cm, and a maximal oxygen uptake (V o 2max) of 47±5 mL/min per kilogram participated in the study. All subjects had a normal ECG and blood pressure and were not taking any medication. The subjects were informed of the risks and discomforts associated with the experiments before giving their informed consent to participate. The study was approved by the Ethical Committee of the Capital Region of Denmark (H-1-2009-081) and conducted in accordance with the guidelines of the declaration of Helsinki. No complications in connection to the invasive procedures were observed.

Experimental Protocol

The subjects completed 5 weeks of 1-legged knee-extensor exercise (3–4 times/week) and 2 weeks of immobilization with the other leg. The subjects were examined on 1 experimental day (experimental protocol 1) before the training/immobilization period and on 2 experimental days (experimental protocol 1 and 2, separated by 2 days) after the training/immobilization period (Figure S1 in the online-only Data Supplement).

Experimental Protocol 1 (Before and After the Training/Immobilization Period)

A microdialysis probe was inserted into the vastus lateralis muscle of the experimental leg(s) under local anesthesia (lidocaine). 23 The subjects completed 10 minutes of 1-legged knee-extensions (24±4 W, ie, 35% of maximal workload (WLmax) before the training/immobilization period). Exercise with the trained and deconditioned leg was separated by 30 minutes of rest and the order was randomized. Muscle dialystate was collected for 10 minutes before the start of exercise, during exercise, and during the recovery from exercise (see Methods in the online-only Data Supplement).

Experimental Protocol 2 (After the Intervention Period)

Three catheters were placed under local anesthesia: A 20-G catheter was inserted into the radial artery of the nondominant arm, a catheter (131HF7, Edwards Lifesciences, Irvine, CA) was inserted in a left antecubital vein and advanced to the pulmonary artery under pressure guidance. A screw-in pacing electrode (Tendril ST, St. Jude Medical, Sylmar, CA) was inserted through the right internal jugular vein and advanced to the right atrium under x-ray guidance, where it was fixated in the atrial wall.

After 30 minutes of supine rest the subjects performed 3 minutes of 1-legged knee-extensor exercise with the (1) deconditioned leg (19±2, 38±4, 56±4 W), (2) trained leg (19±2, 35±4, 56±4, 75±5 W), and (3) trained leg with HR pacing (AAI mode) to elicit the same HR as recorded during exercise with the deconditioned leg (38±4 and 56±4 W Figure S2). Exercise bouts were separated by 10 minutes of seated rest, whereas the 3 trials were separated by 30 minutes of supine rest. Leg blood flow and blood samples (pulmonary and radial artery) were obtained simultaneously before and after 2.5 minutes of exercise.

HR was obtained from an ECG, while mean arterial pressure (MAP), mean pulmonary pressure, pulmonary capillary wedge pressure (PCWP), and central venous pressures (CVP) were monitored with transducers positioned at the level of the heart (Pressure Monitoring Kit, Baxter, IL). The PCWP was determined as at the end of expiration (PCWPend exp). Left ventricular transmural filling pressure was expressed as PCWP minus CVP. 24 Pulmonary V o 2 was measured with a metabolic system (Quark CPET system, Cosmed, Italy). CO was calculated using the Fick equation (CO=V o 2/a-vO2 difference). Femoral arterial blood flow was measured with an ultrasound machine (Philips Ie33, Philips Healthcare, The Netherlands) equipped with a linear probe operating at 5 MHz. Middle cerebral artery velocity was measured by transcranial Doppler (2 MHz) through the temporal ultrasound window at a depth of 48 to 60 mm (Multidop X, DWL, Sipplingen, Germany). Leg mass was calculated from whole-body dual-energy x-ray absorptiometry scanning (Prodigy, General Electrics Medical Systems, WI).

Analytical Procedures

Blood gas variables, hemoglobin, lactate, and K + concentrations and pH were measured using an ABL725 analyzer (Radiometer, Copenhagen, Denmark) and corrected for central venous temperatures. Plasma catecholamines concentrations were determined with a radioimmunoassay (LDN, Nordhorn, Germany). Interstitial ATP, ADP, AMP, and adenosine concentrations were determined by HPLC.

Statistical Analysis

A 2-way repeated measures ANOVA was performed to test statistical significance within and between trials. After a significant F test, pair-wise differences were identified by the Tukey honestly significant difference post hoc procedure. The significance level was set at P<0.05, and data are mean±SEM for 8 subjects unless otherwise indicated.

Results

Performance

WLmax during the incremental 1-legged knee-extensor test was 67±5 W before the intervention period and increased with exercise training to 86±6 W (P<0.05), and was lowered with detraining to 61±4 W (P<0.05). Consequently, the relative workload during the experiment was 31±1%, 62±1%, and 91±1% of WLmax in the detrained leg, and 22±1%, 44±1%, 65±1%, and 87±1% of WLmax in the trained leg.

Systemic Hemodynamics During Exercise With the Trained and Deconditioned Leg

Exercise increased cardiac output (CO) in proportion to the workload and to similar levels during exercise with the trained and deconditioned leg (Figure 1). However, HR was lower when exercise was performed with the trained leg at 38 W (105±3 and 118±3 bpm with the trained and deconditioned leg, respectively) and 58 W (118±4 and 138±6 bpm, respectively P<0.05), whereas stroke volume (SV) was higher (56 W P<0.05). Exercise increased radial and pulmonary arterial blood pressures, but both pressures were lower when exercise was performed with the trained leg (Figures 1 and 2 P<0.05).

Figure 1. Cardiac output, heart rate, stroke volume, and blood pressures at rest and during exercise with a deconditioned and trained leg with and without atrial pacing. Data are mean±SEM. *Different from rest, P<0.05. #Different from deconditioned leg, P<0.05. ¤Different from trained leg without pacing, P<0.05.

Figure 2. Left ventricular performance during exercise with a deconditioned and trained leg, with and without atrial pacing. Note that stroke volume during exercise with the deconditioned and trained leg was coupled to the arterial blood pressure, whereas stroke volume was more coupled to the left ventricular filling pressure (transmural pressure) during atrial pacing. Data are mean±SEM.

During exercise with the trained leg, CVP was reduced compared with baseline (P<0.05), whereas CVP did not change during exercise with the deconditioned leg. PCWP increased from rest to exercise, and there was no difference between values obtained during exercise with the trained and deconditioned leg. The left ventricular contractility index (dP/dtmax) increased similarly during exercise with the trained and deconditioned leg (Figure 3). The rate-pressure product increased during exercise in both conditions (P<0.05), but was lower during exercise (at 38 and 58 W) with the trained leg compared with the deconditioned leg (P<0.05). Pulmonary ventilation, tidal volume, and V co 2 were higher during exercise with the deconditioned leg (P<0.05), whereas the ventilatory frequency was similar (Figure 4). The Ve/V co 2 ratio was lower at 38 W (P<0.05) and tended (P=0.066) to be lower at 19 W, whereas there was no difference at 56 W. The lower exercise induced increase in HR and MAP during exercise with the trained compared with the detrained leg was detectable from 10 seconds after the onset of exercise, but the difference was larger after 30seconds of exercise compared with the initial 30 seconds of exercise (P<0.05 Figure S3).

Figure 3. Left ventricular contractility index (dP/dtmax), arterial plasma norepinephrine (NE), and rate-pressure product at rest and during exercise with a deconditioned and trained leg, with and without atrial pacing. Data are mean±SEM. *Different from rest, P<0.05. #Different from deconditioned leg, P<0.05.

Figure 4. Pulmonary ventilation, respiration frequency, and tidal volume at rest and during exercise with a deconditioned and trained leg with and without atrial pacing. Data are mean±SEM.*Different from rest, P<0.05. #Different from deconditioned leg, P<0.05.

There were no significant differences in blood gas variables, hemoglobin, or O2 content between trials (Table S1). Radial and pulmonary arterial lactate and K + concentrations were higher during exercise with the deconditioned leg (at 38 and 56 W P<0.05), whereas pulmonary arterial pH was lower (at 19 and 38 W P<0.05). Plasma NE concentrations increased during exercise in all 3 trials, but was lower during exercise with the trained leg at 56 W (P<0.05) and tended (P=0.057) to be lower at 38 W. Plasma epinephrine concentrations increased during exercise with the trained leg at 75 W only (P<0.05), but there was no difference between trials.

Effect of Atrial Pacing on Systemic Hemodynamics During Exercise With a Trained Leg

Atrial pacing during exercise with the trained leg increased HR to similar values as during exercise with the deconditioned leg (123±5 [pace] and 119±3 [deconditioned] bpm at 38 W and 141±7 [pace] and 138±6 [deconditioned] bpm at 58 W). CO, SV, CVP, pulmonary arterial, and diastolic blood pressures were similar during the pacing trial and during exercise with the deconditioned leg, whereas systolic blood pressure and MAP were lower.

Compared with exercise with the trained leg without pacing, CO during the pacing trial was similar because of a parallel decrease in SV (P<0.05). Mean arterial and pulmonary pressures, CVP, PCWP, dP/dtmax, and the rate-pressure product were also unchanged, but atrial pacing lowered the systolic pressure (P<0.05). Atrial pacing did not alter any blood gas variables.

Effect of Exercise With a Trained or Deconditioned Leg on Peripheral Hemodynamics

Leg blood flow was lower during exercise with the trained leg compared with exercise with the deconditioned leg at 38 W (P<0.05) and tended (P=0.063) also to be lower at 56 W, whereasthere was no difference in leg vascular conductance between the 2 legs (Figure S4). Middle cerebral artery Vmean increased during exercise at 19 W with both the trained and deconditioned leg, whereas it only tended to increase (P=0.062–0.070) at higher workloads. There was no difference in middle cerebral artery Vmean or cerebral conductance index between when exercise was performed with the trained or deconditioned leg.

Muscle Interstitial Nucleotide and Adenosine Concentrations

Muscle interstitial ATP, ADP, AMP, and adenosine concentrations were similar in the 3 conditions at rest and increased during exercise, and returned to baseline concentrations in the recovery period (Figure 5 and Table S2 P<0.001). The ATP concentration was, however, lower during exercise with the deconditioned muscle compared with exercise with the control and trained muscles (P<0.05), whereas ADP, AMP, and adenosine concentrations were similar. The total interstitial nucleotide/nucleoside concentration was higher in the trained muscle compared with the deconditioned muscle (P<0.05). In the nonexercising muscle, ATP, ADP, AMP, and adenosine concentrations did not change from resting values during exercise with the contralateral leg. Arterial and femoral venous blood pressure, HR, and rate-pressure product from this experimental day are presented in Table S3.

Figure 5. Interstitiel ATP and adenosine concentrations at rest and during exercise and the recovery from exercise in the control, trained, and deconditioned muscle. Data are mean±SEM. *Different from rest P<0.001. §Different from control leg, P<0.001. ¤Different from trained leg, P<0.05.

Discussion

Several findings in this study support an important role of the training status of contracting skeletal muscles for the central hemodynamic response to exercise. First, HR was lower during exercise with the trained leg, whereas CO remained unaltered because of a parallel increase in SV. Second, arterial, pulmonary, and central venous pressures were lower during exercise with the trained leg, whereas PCWP was similar. Third, plasma NE concentrations were lower during exercise with the trained leg. Fourth, interstitial ATP and venous pH were higher during exercise with the trained leg, whereas mixed venous lactate and K + concentrations were lower. Collectively, these results demonstrate that the central response to exercise is tightly coupled to the training status of the contracting skeletal muscle and the relative workload performed and that these changes can occur independently of adaptations within the central circulation and without compromising CO and O2 delivery. Reduced afferent feedback from contracting skeletal muscle resulting in a lower sympathetic nerve activity in the trained state seems to play an important role for the attenuated HR and blood pressure response to exercise. Altered lactate and K + concentrations and pH, but not interstitial nucleotides and adenosine, can contribute to the training-induced attenuated sympathetic activation.

Central Response to Exercise With a Trained and Deconditioned Leg

To differentiate between the contribution of local and central adaptations for the training-induced lowering of HR, we used a set-up that created a large difference in the training status of the leg muscles within the same central circulation. We found that HR, blood pressure, and ventilation were lower when the trained leg was exercising at the same workload, suggesting lower activation of the sympathetic nervous system as confirmed by the attenuated NE response during exercise with the trained leg. 13 These differences were related to the changes within the skeletal muscle that result in an increased WLmax, because the responses were similar when expressed at the same relative workload (Figure 6). Changes in HR and blood pressure during exercise are tightly coupled to improvements in V o 2max and thus relative exercise intensity. 4 Here, we demonstrate that similar changes in SV, MAP, and HR can occur independent of the central adaptations to exercise training (ie, an increase in V o 2max, maximal CO, heart size, left ventricular function, and blood volume). 3,4 Importantly, the attenuated HR and MAP response to exercise did not affect CO, because of a parallel reverse change in SV. The unaltered CO during exercise with a trained and detrained muscle is consistent with the tight coupling between O2 delivery and metabolic demand, 25 but the regulatory mechanisms increasing SV when HR is reduced in the trained state have been unclear. In the trained leg, the higher SV seems to be coupled to a lower cardiac afterload (arterial pressure) during exercise at a given workload, because the left ventricular contractility index and filling pressures were similar when exercise was performed with the trained and deconditioned leg at the same workload. The similar contractility index suggests that myocardial force production during exercise with the trained leg relies on cardiac filing (Frank-Starling mechanism) or altered cardiac cycle length (interval–force relationship). 26 Consequently, the rate-pressure product was lower during exercise with the trained leg, indicating that the myocardial oxygen consumption was reduced by ≈20% during exercise with the trained leg, despite a similar CO. 27

Figure 6. Heart rate, blood pressure, and ventilation at rest and during exercise with a deconditioned and trained leg with and without atrial pacing plotted against the relative workload of the leg. Data are mean±SEM. *Different from rest, P<0.05. ¤Different from trained leg without pacing, P<0.05.

To evaluate the importance of the lower HR response to exercise with the trained leg, HR was increased by atrial pacing during exercise with the trained leg to elicit the same HR as during exercise with the deconditioned leg. The increase in HR did not change CO or arterial pressures, suggesting that the attenuated HR response with exercise training is not of major regulatory consequence and that the difference in blood pressure was not directly coupled to the lower HR. Instead, the lower SV during atrial pacing was related to a reduction in ventricular filling pressure, suggesting that an unaltered venous return was the mechanism by which CO was maintained during the pacing trial. The similar CO and blood pressure levels with and without atrial pacing is in agreement with observations in resting and exercising humans 28 and dogs. 29 Collectively, these observations suggest that CO and O2 delivery are regulated mainly by peripheral O2 demand 28,30 and that the regulation can occur independently of the lower sympathetic activation and consequently HR, blood pressure, and ventilatory response to exercise in the trained state. The lower HR during exercise in the trained state is paralleled by a higher SV that occurs secondary to the reduced cardiac afterload.

Skeletal Muscle Signaling Mechanisms

The tight coupling between the relative load on the skeletal muscles and cardioventilatory response demonstrates that the training status of the skeletal muscles was driving these changes, but the underlying signaling mechanisms are likely to include several factors. First, exercise with the trained leg was associated with lower mixed venous K + and lactate concentrations and a higher pH. Interstitial lactate concentrations increase with exercise intensity 16 and blockade of acid sensitive ion channels attenuates the pressor response in cats. 31 In support of a role of K + and pH for the exercise pressor reflex, a relationship between K + concentrations and MAP 15 and between muscle pH and sympathetic activity has been reported. 32 Second, differences in central command are also likely to have contributed to the observed change in responses due to differences in the relative exercise intensity between the 2 legs 10 and consequently increased motor unit recruitment and perceived exertion. Third, differences in circulating substances released from skeletal muscle may also have contributed by stimulating peripheral chemoreceptors. Also, the higher pH and lower P co2 levels may have contributed to the lower ventilation during exercise with the trained leg. Last, increased venous distension coupled to the higher femoral venous blood pressures in the exercising detrained leg may also have contributed by increasing afferent signaling. 33 Although the relative contribution of each of these variables is unclear, the difference in HR and MAP during exercise with the 2 exercising legs was delayed such that it was larger after 30 seconds of exercise compared with at the onset of exercise. Group IV afferent fibers show a 5- to 15-second delay in response from the onset of exercise reflecting their activation by metabolic substances. 10,12 Altered afferent feedback therefore seems to account for part of the observed differences in cardioventilatory response.

The increase in muscle interstitial ATP concentrations during exercise was markedly lower in the deconditioned muscle. The lower interstitial ATP concentrations but higher MAP, HR, and plasma NE concentration during exercise with the deconditioned leg suggest that ATP alone or in synergy with other substances 34 is not an obligatory mediator of the training-induced changes in afferent signaling. ATP is released from contracting skeletal muscle cells 35 as well as from endothelial cells in response to mechanical stress. 36 The lower increase in total adenine nucleotides and adenosine in the deconditioned leg suggests that deconditioning lowered the release of ATP into the interstitial space rather than a lower resynthesis of ATP in the interstitial space from its breakdown products.

ATP can override sympathetic vasoconstrictor activity (functional sympatholysis) in a similar manner as exercise. 37,38 In the same subjects, we have found a coupling between the muscle training status and the degree of functional sympatholysis during exercise. 39 The sympatholytic properties of ATP are not affected by the training status of the skeletal muscles. 23,39 The lower interstitial ATP concentrations and impaired functional sympatholysis in the deconditioned leg is in agreement with the observation that interstital ATP concentrations and the degree of functional sympatholysis during exercise is lower in sedentary elderly compared with young and trained elderly. 23 Taken together, the coupling between changes in interstitial ATP levels and the degree of functional sympatholysis during exercise observations open up for the possibility that the interstitial ATP plays a role in mediating functional sympatholysis during exercise, and thereby contributes by optimizing the blood flow distribution within the contracting muscle and the lowering of leg blood flow with exercise training. 23,40,41 Although the physiological role of ATP-sensitive P2Y2 receptors on smooth muscle cells of human skeletal muscle 42 remain undisclosed, the presence of these receptors and the relatively high interstitial ATP levels as compared with plasma ATP levels 43 suggest a functional role of interstitial ATP in the regulation of the vascular tone.

Conclusion

The lower HR, blood pressure, and ventilatory responses to exercise at a given workload with a trained skeletal muscle suggest that factors within the contracting skeletal muscle contribute to a lower sympathetic activation during exercise. Changes in skeletal muscle lactate and K + concentrations and pH are likely to contribute to these changes by altering afferent feedback, whereas the markedly lower interstitial ATP concentrations during exercise with the previously immobilized leg suggest that interstitial ATP contributes to blood flow regulation in other ways than by simulating muscle afferents. The similar CO and O2 delivery, despite an 8% to 14% lower HR and blood pressure during exercise with the trained leg, suggest that adaptations within the skeletal muscles can result in ≈20% lower myocardial work during exercise without compromising O2 delivery and aerobic metabolism.

Perspective

In cardiovascular diseases associated with an impaired muscle perfusion, the exercise pressor reflex is augmented 44 and physical inactivity is therefore likely to aggravate the sympathetic response to exercise. In chronic heart failure, excessive ventilation coupled to increased skeletal muscle signaling impairs exercise tolerance. 45,46 Previous studies have demonstrated that single leg training can improve peak leg V o 2 and exercise capacity in patients, and single leg training is a well-tolerated exercise model. 47–49 Exercise training with 1 leg at a time can therefore be a useful intervention to lower the blood pressure, HR, and ventilatory response during exercise, improve skeletal muscle tissue perfusion and metabolism and, consequently, lower the load on the heart during daily physical activities while avoiding the acute strain on the central circulation associated with whole-body exercise.

Acknowledgments

The skilful technical assistance of Karina Olsen is gratefully acknowledged.

Sources of Funding

This study was supported by a grant from the Lundbeck foundation . Dr Mortensen was supported by a grant from the Danish Heart Foundation and The Danish Council for Independent Research-Medical Sciences .