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In 2012, I read an article by Grassi et al. (Faster O2 uptake kinetics in canine muscle in situ after acute creatine kinase inhibition. J Physiol. 2011; 589(1):221-233), and was astonished by the numerous violations of quality science in the methods and data interpretation. Yes, these are experienced researchers, but that does not entitle authority to take short cuts in science, or receive special treatment in editorial peer review, as these events in turn can only undermine science. In this case, the topic in science was skeletal muscle energy metabolism during exercise; a topic that I am more than qualified to provide critical commentary on. Strangely, this manuscript was followed by a letter to the editor by Poole D.C. (Oxygen's double edged sword: balancing muscle O2 supply and use during exercise. 2011; 589(3):457-458) where support of the research methods and results of Grassi et al. were exalted, with Poole stating "The elegant CK blockade .....". Surely an experienced editor "should smell a rat" when science deviates from critical challenge to affirmations of support? What is the professional association and potential bias between these authors?
I then wrote a letter to the editor, followed by a short commentary. Despite all my arguments and explanations, which were all supported by empirical evidence and fact, my criticisms went unsupported. This was in part due to a highly biased review, where the editor of the journal at question obviously thought it was not a violation of bias in peer review to have Grassi (the lead author of the manuscript in question) review my criticism and recommend whether or not it be published! Can you believe that? Things went from bad to worse over this issue. I will not provide too much detail here, as I am still trying to publish the commentary. However, I will summarize the study by Grassi et al., and then present some of my pertinent comments and explanations for why this study was so off mark, and how it started my research journey into VO2 kinetics to steady state.
Grassi et al. researched the influence of acute iodoacetamide (IA) inhibition of creatine kinase (CK) in isolated in-situ canine skeletal muscle. Isometric muscle contractions for rest to exercise transitions to steady state were elicited by electrical stimulation (200 ms contraction followed by 1.3 s passive recovery) to cause a metabolic intensity equivalent to approximately 70% muscle VO2 peak. During the IA trial condition, IA was infused over a 10 min pre-contraction period to establish a stable blood IA concentration of approximately 5.0 mmol/L. Arterial and venous blood samples, as well as muscle biopsy samples were obtained to quantify the extent of metabolic perturbation for control and IA conditions.
The figure (manuscript Figure 2, p. 227) on the left reveals the change in muscle ATP and creatine phosphate (CrP = PCr) for the control vs. IA conditions pre-exercise (rest) and during contractions at steady state (contractions). Note the appreciable decline in muscle ATP pre-contraction for the IA trial. In short, the IA infusion caused severe muscle poisoning under rest (low ATP turnover) conditions. IA infusion was effective in causing a stable muscle CrP concentration. However, as is expected, the IA caused large declines in both contractile force and VO2, as shown in the second figure below left (manuscript Figure 1, p. 226). In this regard, the research of Harrison et al. (CK inhibition accelerates transcytosolic energy signaling during rapid workload steps in isolated rabbit hearts) is pertinent. These researchers studied the inhibitory function of IA on multiple enzymes of cellular energy catabolism. Their results were clear in showing how IA not only inhibits CK, but also adenylate kinase, glyceraldehyde-3-phosphate dehydrogenase, and posssibly also phosphorylase based on the similarity in structure and function between IA and iodoacetate; a known inhibitor of phosphorylase. Furthermore, such inhibition was documented for circulatory IA concentrations of 0.4 mmol/L; far lower than the 5 mmol/L used by Grassi et al.! In short, IA is not a selective and specific inhibitor of CK. Rather, IA inhibits numerous enzymes of the phosphagen and glycolytic energy systems, effectively compromising non-mitochondrial ATP turnover, and thereby severely poisoning skeletal muscle. This is far from the "elegant CK blockade" expressed by Poole!
The added data on the left reveal the extent of the catabolic perturbation by IA (Figure 1, page 226), causing large reductions in contractile force, and almost a 60% reduction in VO2. The bottom figure on the left is where my discontent with current methods and interpretation in VO2 kinetics to steady state began. Grassi et al. argued that because of first order linear kinetics, it is valid to interpret the kinetic response in VO2 between two conditions, regardless of the magnitude of the VO2 response or any other difference that could influence cellular respiration (see content below on cellular ATP, ADP and Pi). To wrap my head around this concept, I read the entire edited text of Poole DC and Jones AM (Oxygen uptake kinetics in sport, exercise and medicine), which provides a thorough review and explanation of prior research and the theory of methodological approaches to the study of VO2 kinetics to steady state prior to 2004. Linear first order kinetics implies that the time constant is invariant across different VO2 responses, regardless of magnitude. I also retrieved most of the key original research articles, and soon discovered more disturbing traits of the field of VO2 kinetics research. More about this journey a bit later into this story.
So, back to the Grassi et al. study. Grassi et al. interpreted their results to reveal that mono-exponential modelling of the VO2 data revealed a larger time constant for the control condition than the IA condition (19.7 vs. 7.2 s, respectively). Note that the modified control trial data was presented to account for the gradual change in IA concentrations, though this data is largely overlooked in the Discussion. Clearly, the basis for the data interpretation rested solely with the concept of linear first order kinetics, combined with the belief that IA provides a CK specific inhibition; which as I have explained it does not.
Yet the data for muscle ATP presented above clearly revealed a disturbing feature of the CK inhibition; a large reduction in muscle ATP at rest and during contractions. For muscle ATP to decline, there has to be a major perturbation to adenylate energy transfer, which would cause appreciable increases in muscle ADP, a potent stimulator of mitochondrial respiration. In addition, along with increased muscle ADP, free inorganic phophate (Pi) would also increase, which in turn can explain the dramatic decline in both muscle contractile force and VO2 due to interference with intracellular calcium flux. Also be aware that Pi is a substrate for oxidative phosphorylation, and an increasing cytosolic Pi will favor increased VO2 kinetics.
Application of the creatine kinase equilibrium (KCK) can estimate the increased ADP for known changes in other substrates and products of the creatine kinase reaction. The KCK for skeletal muscle can be calculated from the following equation for the conditions of temperature = 38 C, free muscle Mg^+2 = 0.001 M/L, and ionic strength = 0.25. Note, all concentrations are expressed M/L of muscle water, and the value for KCK is based on the work of Lawson and Veech (J Biol Chem. 264:6528-6537, 1979).
Resting Muscle
KCK = 1.66 x 10^9
= ([ATP] [Cr]) / ([ADP] [CrP] [H+])
= (0.0095 x 0.005) / ([ADP] x 0.033 x 0.0000001)
[ADP] = 8.67 x 10^-6 M/L or 8.67 umol/L
It is difficult to really know the muscle ADP concentration, as this value is too low for measurement. Theoretical computation of the KCK as shown above reveals a value of 8.67 umol/L. Again, be aware that these calculations are for muscle at rest.
Extreme Exercise
For extreme exercise conditions, the above computations change in accord with the different cellular conditions for all metabolites. We can estimated with some degree of accuracy what all components are, except again for the ADP, which is solved once again by using the KCK (Note that there is error here, as the KCK is not really a constant given that it changes with different metabolic states of an active tissue).
KCK = 1.66 x 10^9
= ([ATP] [Cr]) / ([ADP] [CrP] [H+])
= (0.005 x 0.03) / ([ADP] x 0.005 x 0.000001)
[ADP] = 1.807 x 10^-4 M/L or 181 umol/L
The point of all this is to show that for extreme muscle contractions to contractile failure, as was induced by the experimental conditions of Grassi et al., intramuscular [ADP] would have increased far higher than the ~21 fold increase shown above. When combined with the increased [Pi], there would have been extreme stimulation for mitochondrial respiration in the IA condition, and this would have been absent in the control condition. Clearly, the inhibition of CK was not the only difference between experimental conditions.
Upon reflection, the exact opposite interpretation is needed for the research of Grassi et al. Given the relative lack of ADP and Pi stimulation of cellular respiration in the control trial, and given the higher absolute kinetics of the initial slope of the VO2 response in the control trial, and given the near three-fold larger delta VO2 of the control trial, indeed, an intact and functional CK enzyme is clearly needed to support rapid increases in cellular VO2. There is no evidence at all for a buffering of cellular ADP by the CK reaction, which in turn desensitizes cellular respiration from ATP demand.
The model of the CK shuttle above is a logical illustration of the need for CK to be functional to optimize intracellular ADP provision to mitochondria. Myofibrillar ATP hydrolysis provides ADP distant to the mitochondria. The rapid shuttling of this ADP through multiple terminal phosphate transfer CK reactions, finally interacting with CKmito allows the release of ADP within the mitochondria and for its availability as a substrate for mitochondrial respiration. The CK shuttle is evidence for the need of a functional CK enzyme to allow rapid terminal phosphate transfer within a metabolically active tissue, such as skeletal muscle.
So why did I have to go through all of this to explain my recent interest in VO2 kinetics to steady state? Grassi et al. relied on the theory of linear first order kinetics for the VO2 transition to steady state. This in turn is dependent on a mono-exponential model to derive the time constant. As early as the 1980's, evidence existed to refute the concept of linear first order VO2 kinetics to steady state. In addition, the errors of the Grassi et al. manuscript make it very clear that if you are over-reliant on a theory that has a dubious base of support, then you compound the errors by incorrect added interpretations of results, which in turn can derail the discipline of VO2 kinetics to steady state and all other applications of this method, which for the Grassi et al. manuscript was to advocate incorrect interpretations of skeletal muscle cellular energy catabolism.
The more I have read on the topics of VO2 kinetics to steady state, the less convinced I am of a sound discipline fueled by balanced and unbiased research. Such revelations led to my 2013 manuscript on the critical review of this discipline (A critical review of the history of low- to moderate intensity steady state VO2 kinetics).
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