Altered Energetics of Exercise Explain Risk of Rhabdomyolysis in Very Long-Chain Acyl-CoA Dehydrogenase Deficiency.

Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD.

Combined in vivo and in silico investigations of activation of glycolysis in contracting skeletal muscle.

The hypothesis was tested that the variation of in vivo glycolytic flux with contraction frequency in skeletal muscle can be qualitatively and quantitatively explained by calcium-calmodulin activation of phosphofructokinase (PFK-1). Ischemic rat tibialis anterior muscle was electrically stimulated at frequencies between 0 and 80 Hz to covary the ATP turnover rate and calcium concentration in the tissue. Estimates of in vivo glycolytic rates and cellular free energetic states were derived from dynamic changes in intramuscular pH and phosphocreatine content, respectively, determined by phosphorus magnetic resonance spectroscopy ((31)P-MRS). Computational modeling was applied to relate these empirical observations to understanding of the biochemistry of muscle glycolysis. Hereto, the kinetic model of PFK activity in a previously reported mathematical model of the glycolytic pathway (Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. J Physiol 588: 1961-1983, 2010) was adapted to contain a calcium-calmodulin binding sensitivity. The two main results were introduction of regulation of PFK-1 activity by binding of a calcium-calmodulin complex in combination with activation by increased concentrations of AMP and ADP was essential to qualitatively and quantitatively explain the experimental observations. Secondly, the model predicted that shutdown of glycolytic ATP production flux in muscle postexercise may lag behind deactivation of PFK-1 (timescales: 5-10 s vs. 100-200 ms, respectively) as a result of accumulation of glycolytic intermediates downstream of PFK during contractions.

Unchanged muscle fiber conduction velocity relates to mild acidosis during exhaustive bicycling.

Muscle fiber conduction velocity (MFCV) has often been shown to decrease during standardized fatiguing isometric contractions. However, several studies have indicated that the MFCV may remain constant during fatiguing dynamic exercise. It was investigated if these observations can be related to the absence of a large decrease in pH and if MFCV can be considered as a good indicator of acidosis, also during dynamic bicycle exercise. High-density surface electromyography (HDsEMG) was combined with read-outs of muscle energetics recorded by in vivo (31)P magnetic resonance spectroscopy (MRS). Measurements were performed during serial exhausting bouts of bicycle exercise at three different workloads. The HDsEMG recordings revealed a small and incoherent variation of MFCV during all high-intensity exercise bouts. (31)P MRS spectra revealed a moderate decrease in pH at the end of exercise (~0.3 units down to 6.8) and a rapid ancillary drop to pH 6.5 during recovery 30 s post-exercise. This additional degree of acidification caused a significant decrease in MFCV during cycling immediately after the rest period. From the data a significant correlation between MFCV and [H(+)] ([H(+)] = 10(-pH)) was calculated (p < 0.001, Pearson's R = -0.87). Our results confirmed the previous observations of MFCV remaining constant during fatiguing dynamic exercise. A constant MFCV is in line with a low degree of acidification, considering the presence of a correlation between pH and MFCV after further increasing acidification.

Increased mitochondrial content rescues in vivo muscle oxidative capacity in long-term high-fat-diet-fed rats.

Mitochondria are thought to play a crucial role in the etiology of muscle insulin resistance (IR). The aim of this study was to gain more insight into the timing and nature of mitochondrial adaptations during the development of high-fat-diet (HFD)-induced IR. Adult Wistar rats were fed HFD or normal chow for 2.5 and 25 wk. Intramyocellular lipids (IMCLs) were quantified in vivo using (1)H magnetic resonance spectroscopy (MRS). Muscle oxidative capacity was assessed in vivo using (31)P MRS and in vitro by measuring mitochondrial DNA copy number and oxygen consumption in isolated mitochondria. MRS in tibialis anterior muscle revealed 3.3-fold higher IMCL content and 1.2-fold increased oxidative capacity after 2.5 wk of HFD feeding. The latter result could be fully accounted for by increased mitochondrial content. After 25 wk of HFD, maximal ADP-stimulated oxygen consumption in isolated mitochondria oxidizing pyruvate plus malate remained unaffected, while IMCL and mitochondrial content had further increased compared to controls (5.1-fold and 1.4-fold, respectively). Interestingly, in vivo oxidative capacity at this time point was identical to controls. These results show that skeletal muscle in HFD-induced IR accompanied by IMCL accumulation requires a progressively larger mitochondrial pool size to maintain normal oxidative capacity in vivo.

Computational modelling identifies the impact of subtle anatomical variations between amphibian and mammalian skeletal muscle on spatiotemporal calcium dynamics.

The physical sites of calcium entry and exit in the skeletal muscle cell are distinct and highly organised in space. It was investigated whether the highly structured spatial organisation of sites of Ca(2+) release, uptake and action in skeletal muscle cells substantially impacts the dynamics of cytosolic Ca(2+) handling and thereby the physiology of the cell. Hereto, the spatiotemporal dynamics of the free calcium distribution in a fast-twitch (FT) muscle sarcomere was studied using a reaction-diffusion computational model for two genotypes with known anatomical differences. A computational model of a murine FT muscle sarcomere is developed, de novo including a closed calcium mass balance to simulate spatiotemporal high stimulation frequency calcium dynamics at 35 degrees C. Literature data on high-frequency calcium dye measurements were used as a first step towards model validation. The murine and amphibian sarcomere models were phenotypically distinct to capture known differences in positions of troponin C, actin-myosin overlap and calcium release within the sarcomere between frog and mouse. The models predicted large calcium gradients throughout the myoplasm as well as differences in calcium concentrations near the mitochondria of frog and mouse. Furthermore, the predicted Ca(2+) concentration was high at positions where Ca(2+) has a regulatory function, close to the mitochondria and troponin C.

Treadmill but not wheel running improves fatigue resistance of isolated extensor digitorum longus muscle in mice.

AIM: The present study is the first to compare the physiological impact of either forced treadmill or voluntary wheel running exercise on hindlimb muscle in mice. METHODS: Male C57BL/6 mice were subjected to either 6 weeks of forced treadmill or voluntary wheel running exercise. Mice in the treadmill running exercise group (TRE; n = 8) ran 1.9 km day(-1) at a speed of 16 m min(-1) against an uphill incline of 11 degrees. In the running wheel exercise group (RWE; n = 8) animals ran 8.8 +/- 0.2 km per day (average speed 42 +/- 2 m min(-1)). After the experimental period, animals were killed and mechanical performance and oxygen consumption of isolated extensor digitorum longus (EDL) muscle were determined during serial electrical stimulation at 0.5, 1 and 2 Hz. RESULTS: Steady-state half-width time (HWT) of twitch contraction at 0.5 Hz was significantly shorter in TRE and RWE than controls (CON) (41.3 +/- 0.2, 41.3 +/- 0.1 and 44.3 +/- 0.1 s respectively; P < 0.05). The rate of fatigue development and HWT lengthening at 2 Hz was the same in RWE and CON but lower in TRE (1.2-fold and twofold respectively; P < 0.05). EDL oxygen consumption, mitochondrial content and myosin heavy chain (MyHC) composition were not different between the groups. CONCLUSION: These results indicate that both exercise modalities have an effect on a hindlimb fast-twitch muscle in mice, with the greatest impact seen with forced treadmill running.

Impaired in vivo mitochondrial function but similar intramyocellular lipid content in patients with type 2 diabetes mellitus and BMI-matched control subjects.

AIMS/HYPOTHESIS: Mitochondrial dysfunction and increased intramyocellular lipid (IMCL) content have both been implicated in the development of insulin resistance and type 2 diabetes mellitus, but the relative contributions of these two factors in the aetiology of diabetes are unknown. As obesity is an independent determinant of IMCL content, we examined mitochondrial function and IMCL content in overweight type 2 diabetes patients and BMI-matched normoglycaemic controls. METHODS: In 12 overweight type 2 diabetes patients and nine controls with similar BMI (29.4 +/- 1 and 29.3 +/- 0.9 kg/m(2) respectively) in vivo mitochondrial function was determined by measuring phosphocreatine recovery half-time (PCr half-time) immediately after exercise, using phosphorus-31 magnetic resonance spectroscopy. IMCL content was determined by proton magnetic resonance spectroscopic imaging and insulin sensitivity was measured with a hyperinsulinaemic-euglycaemic clamp. RESULTS: The PCr half-time was 45% longer in diabetic patients compared with controls (27.3 +/- 3.5 vs 18.7 +/- 0.9 s, p < 0.05), whereas IMCL content was similar (1.37 +/- 0.30 vs 1.25 +/- 0.22% of the water resonance), and insulin sensitivity was reduced in type 2 diabetes patients (26.0 +/- 2.2 vs 18.9 +/- 2.3 mumol min(-1) kg(-1), p < 0.05 [all mean +/- SEM]). PCr half-time correlated positively with fasting plasma glucose (r (2) = 0.42, p < 0.01) and HbA(1c) (r (2) = 0.48, p < 0.05) in diabetic patients. CONCLUSIONS/INTERPRETATION: The finding that in vivo mitochondrial function is decreased in type 2 diabetes patients compared with controls whereas IMCL content is similar suggests that low mitochondrial function is more strongly associated with insulin resistance and type 2 diabetes than a high IMCL content per se. Whether low mitochondrial function is a cause or consequence of the disease remains to be investigated.

Computational model of excitable cell indicates ATP free energy dynamics in response to calcium oscillations are undampened by cytosolic ATP buffers.

Mitochondria in excitable cells are recurrently exposed to pulsatile calcium gradients that activate cell function. Rapid calcium uptake by the mitochondria has previously been shown to cause uncoupling of oxidative phosphorylation. To test (i) if periodic nerve firing may cause oscillation of the cytosolic thermodynamic potential of ATP hydrolysis and (ii) if cytosolic adenylate (AK) and creatine kinase (CK) ATP buffering reactions dampen such oscillations, a lumped kinetic model of an excitable cell capturing major aspects of the physiology has been developed. Activation of ATP metabolism by low-frequency calcium pulses caused large oscillation of the cytosolic, but not mitochondrial ATP/ADP, ratio. This outcome was independent of net ATP synthesis or hydrolysis during mitochondrial calcium uptake. The AK/CK ATP buffering reactions dampened the amplitude and rate of cytosolic ATP/ADP changes on a timescale of seconds, but not milliseconds. These model predictions suggest that alternative sources of capacitance in neurons and striated muscles should be considered to protect ATP-free energy-driven cell functions.

Robust homeostatic control of quadriceps pH during natural locomotor activity in man.

It is generally thought that intracellular pH (pHi) of skeletal muscle falls at least 0.5 units during intense activity, but evidence on natural (i.e., voluntary, two-legged (2L)) locomotor activity in man has exclusively come from invasive studies of upper leg muscle. Here, noninvasive (31)P nuclear magnetic resonance spectroscopy ((31)P NMRS) was used to study human quadriceps muscle energetics and pHi during incremental bicycling exercise to exhaustion in six normally active subjects. Cellular energy charge (CEC; [PCr]/([PCr]+[Pi])) linearly (r 0.90) dropped 83 +/- 3% during ramp exercise to exhaustion from 0.92 +/- 0.01 at rest to 0.16 +/- 0.03 at maximal sustained work rate (WR) (166+/-17 W; range: 108-223 W). Surprisingly, pHi likewise dropped linearly (r 0.82) no more than 0.2 units over the entire range of WR between rest and maximal (pHi 7.08+/-0.01 and 6.84+/-0.02, respectively). But after termination of exercise pHi dropped rapidly to textbook acidic values of 6.6 explaining classic biopsy results. Comparative coresponse analysis of pHi and CEC changes during 2L- vs. 1L-cycling showed that homeostatic control of quadriceps pHi during bicycling is robust and unique to natural locomotor exercise. These results highlight the robustness of the integrative set of physicochemical and physiological control mechanisms in acid-base balance during natural locomotor activity in man.

Regulation analysis of contractile ATPase flux in skeletal muscle.

J The [Ca2+] regulation of contractile ATPase flux, Jp, in skeletal muscle was analysed by computation of the Response R(Jp) Ca2+ for a 10 Hz range of electrical stimulation frequencies. Results of our analysis of the kinetic controls in ATP free energy metabolism in a network model of contracting muscle (J.A.L. Jeneson, H.V. Westerhoff and M.J. Kushmerick (2000) Am. J Physiol. 279, C813-C832) formed the basis for the computations of R(Jp) Ca2+. We found that neural regulation of sustained force generation via simple [Ca2+]cyto frequency encoding in the network was robust for frequencies up to 2 Hz. Above 2 Hz, however, this regulation design broke down because of a shift in contractile ATPase flux control from the Ca2+-sensitive contractile filaments to mitochondria with low Ca2+ sensitivity. The role of glyco(geno)lytic ATP production at high contraction workloads is discussed in the context of this result.

Resting oxygen consumption and in vivo ADP are increased in myopathy due to complex I deficiency.

BACKGROUND: Patients with isolated complex I deficiency (CID) in skeletal muscle mitochondria often present with exercise intolerance as their major clinical symptom. OBJECTIVE: To study the in vivo bioenergetics in patients with complex I deficiency in skeletal muscle mitochondria. METHODS: In vivo bioenergetics were studied in three of these patients by measuring oxygen uptake at rest and during maximal exercise, together with forearm ADP concentrations ([ADP]) at rest. Whole-body oxygen consumption at rest (VO(2)) was measured with respiratory calorimetry. Maximal oxygen uptake (VO(2)max) was measured during maximal exercise on a cycle ergometer. Resting [ADP] was estimated from in vivo (31)P MRS measurements of inorganic phosphate, phosphocreatine, and ATP content of forearm muscle. RESULTS: Resting VO(2) was significantly increased in all three patients: 128 +/- 14% (SD) of values in healthy control subjects. VO(2)max in patients was on average 2.8 times their VO(2) at rest and was only 28% of VO(2)max in control subjects. Resting [ADP] in forearm muscle was significantly increased compared with healthy control subjects (patients 26 +/- 2 microM, healthy controls 9 +/- 2 microM). CONCLUSION: In patients with CID, the increased whole-body oxygen consumption rate at rest reflects increased electron transport through the respiratory chain, driven by a decreased phosphorylation potential. The increased electron transport rate may compensate for the decreased efficiency of oxidative phosphorylation (phosphorylation potential).