In vivo ATP production during free-flow and ischaemic muscle contractions in humans

Abstract

The aim of this study was to determine how ATP synthesis and contractility in vivo are altered by ischaemia in working human skeletal muscle. The hypotheses were: (1) glycolytic flux would be higher during ischaemic (ISC) compared to free-flow (FF) muscle contractions, in compensation for reduced oxidative ATP synthesis, and (2) ischaemic muscle fatigue would be related to the accumulation of inhibitory metabolic by-products rather than to the phosphorylation potential ([ATP]/[ADP][P(i)]) of the muscle. Twelve healthy adults (6 men, 6 women) performed six intermittent maximal isometric contractions of the ankle dorsiflexors (12 s contract, 12 s relax), once with intact blood flow and once with local ischaemia by thigh cuff inflation to 220 Torr. Intracellular phosphorous metabolites and pH were measured non-invasively with magnetic resonance spectroscopy, and rates of ATP synthesis through oxidative phosphorylation, anaerobic glycolysis, and the creatine kinase reaction were determined. The force-time integral declined more during ISC (66 +/- 3% initial) than FF (75 +/- 2% initial, P = 0.002), indicating greater fatigue in ISC. [ATP] was preserved in both protocols, indicating matching of ATP production and use under both conditions. Glycolytic flux (mm s(-1)) was similar during FF and ISC (P = 0.16). Total ATP synthesis rate was lower during ISC, despite adjustment for the greater muscle fatigue in this condition (P < 0.001). Fatigue was linearly associated with diprotonated inorganic phosphate (FF r = 0.94 +/- 0.01, ISC r = 0.92 +/- 0.02), but not phosphorylation potential. These data provide novel evidence that ATP supply and demand in vivo are balanced in human skeletal muscle during ischaemic work, not through higher glycolytic flux, but rather through increased metabolic economy and decreased rates of ATP consumption as fatigue ensues.

Figure 1. ³¹P MRS spectra from the tibialis anterior muscle in a single subject

Representative stacked plot of phosphorous spectra at rest (first spectrum, 60 s average) and throughout the six 12 s MVCs with 12 s rest intervals (4 s temporal resolution). The P_i peak split into two distinct peaks during the contractions in this subject.

Figure 2. Muscle fatigue of the ankle dorsiflexors

The force–time integral during the 12 s MVCs, expressed relative to the first contraction, with (ISC, ○) and without (FF, •) local circulatory occlusion. There was more fatigue during ISC compared to FF (P < 0.001). Values are means ± s.e.m.

Figure 3. Phosphocreatine and pH during contractions and recovery

Phosphocreatine (A) decreased more during ISC (○) compared to FF (•) (P < 0.001). The inset shows the contraction period, illustrating transient PCr depletion and recovery during the contraction–relaxation cycle. Intracellular pH (B) decreased during FF and ISC, with a trend (P = 0.10) for greater acidosis during ISC. Values are means ±s.e.m.

Figure 4. Rates of ATP synthesis during muscle contractions

ATP production from oxidative phosphorylation (ATP_OX, A) was similar across the 6 contractions throughout FF (P = 0.12), and assumed to be negligible during ISC. Glycolytic flux (ATP_GLY, B) was similar during FF (•) and ISC (○) (P = 0.22). ATP production from net PCr breakdown in the creatine kinase reaction (ATP_CK, C) was greater during FF than ISC (P < 0.001). Total ATP turnover (ATP_TOT, D), calculated as the sum of ATP_OX, ATP_GLY, and ATP_CK, was greater during FF compared to ISC (P < 0.001). Values are means ±s.e.m.

Figure 5. Relationships between muscle fatigue, pH and phosphorylation potential

Strong linear associations between fatigue and H₂PO₄⁻ were observed during FF (•) and ISC (○) (top panel); FF r = 0.93 ± 0.01, ISC r = 0.91 ± 0.03. Curvilinear relationships were evident between fatigue and intracellular phosphorylation potential during FF and ISC (bottom panel). These relationships were left-shifted during ISC compared to FF. Values are means ±s.e.m.

Figure 6. ¹H-MRS measures of deoxy-myoglobin during ischaemic contractions in one subject

The deoxy-myoglobin signal was measured during 1 min of resting cuff occlusion (○, ischaemia), ISC protocol (contraction period defined by the dotted vertical lines), cuff occlusion for 1 min following the end of the contractions, and 10 min of free-flow recovery (•, release of cuff occlusion). Ischaemic muscle contractions resulted in myoglobin desaturation to ∼75% of the desaturation observed during 10 min of resting ischaemia.

Figure 7. ³¹P-MRS measures of phosphocreatine during prolonged ischaemia followed by ischaemic contractions in one subject

PCr decreased slightly after ∼8 min of resting ischaemia (○, ischaemia). Muscle contractions were initiated after 10 min of ischaemia, resulting in a marked decrease in PCr (the start and end of the contraction periods are indicated by the dotted lines). Inset shows PCr during 6 MVC/rest intervals. PCr recovery was observed following the first 3 contractions. No recovery of PCr was evident when ischaemia was maintained for 1 min after the final MVC, but PCr recovered quickly when blood flow was restored (•, release of cuff occlusion).