Youngran Chung

Comparative analysis of NMR and NIRS measurements of intracellular PO2 in human skeletal muscle.

1H NMR has detected both the deoxygenated proximal histidyl NδH signals of myoglobin (deoxyMb) and deoxygenated Hb (deoxyHb) from human gastrocnemius muscle. Exercising the muscle or pressure cuffing the leg to reduce blood flow elicits the appearance of the deoxyMb signal, which increases in intensity as cellular[Formula: see text] decreases. The deoxyMb signal is detected with a 45-s time resolution and reaches a steady-state level within 5 min of pressure cuffing. Its desaturation kinetics match those observed in the near-infrared spectroscopy (NIRS) experiments, implying that the NIRS signals are actually monitoring Mb desaturation. That interpretation is consistent with the signal intensity and desaturation of the deoxyHb proximal histidyl NδH signal from the β-subunit at 73 parts per million. The experimental results establish the feasibility and methodology to observe the deoxyMb and Hb signals in skeletal muscle, help clarify the origin of the NIRS signal, and set a stage for continuing study of O2regulation in skeletal muscle.1H NMR has detected both the deoxygenated proximal histidyl NdeltaH signals of myoglobin (deoxyMb) and deoxygenated Hb (deoxyHb) from human gastrocnemius muscle. Exercising the muscle or pressure cuffing the leg to reduce blood flow elicits the appearance of the deoxyMb signal, which increases in intensity as cellular PO2 decreases. The deoxyMb signal is detected with a 45-s time resolution and reaches a steady-state level within 5 min of pressure cuffing. Its desaturation kinetics match those observed in the near-infrared spectroscopy (NIRS) experiments, implying that the NIRS signals are actually monitoring Mb desaturation. That interpretation is consistent with the signal intensity and desaturation of the deoxyHb proximal histidyl NdeltaH signal from the beta-subunit at 73 parts per million. The experimental results establish the feasibility and methodology to observe the deoxyMb and Hb signals in skeletal muscle, help clarify the origin of the NIRS signal, and set a stage for continuing study of O2 regulation in skeletal muscle.

Myoglobin desaturation with exercise intensity in human gastrocnemius muscle

The present study evaluated whether intracellular partial pressure of O(2) (PO(2)) modulates the muscle O(2) uptake (VO(2)) as exercise intensity increased. Indirect calorimetry followed VO(2), whereas nuclear magnetic resonance (NMR) monitored the high-energy phosphate levels, intracellular pH, and intracellular PO(2) in the gastrocnemius muscle of four untrained subjects at rest, during plantar flexion exercise with a constant load at a repetition rate of 0.75, 0.92, and 1.17 Hz, and during postexercise recovery. VO(2) increased linearly with exercise intensity and peaked at 1.17 Hz (15. 1 +/- 0.37 watts), when the subjects could maintain the exercise for only 3 min. VO(2) reached a peak value of 13.0 +/- 1.59 ml O(2). min(-1). 100 ml leg volume(-1). The (31)P spectra indicated that phosphocreatine decreased to 32% of its resting value, whereas intracellular pH decreased linearly with power output, reaching 6.86. Muscle ATP concentration, however, remained constant throughout the exercise protocol. The (1)H NMR deoxymyoglobin signal, reflecting the cellular PO(2), decreased in proportion to increments in power output and VO(2). At the highest exercise intensity and peak VO(2), myoglobin was approximately 50% desaturated. These findings, taken together, suggest that the O(2) gradient from hemoglobin to the mitochondria can modulate the O(2) flux to meet the increased VO(2) in exercising muscle, but declining cellular PO(2) during enhanced mitochondrial respiration suggests that O(2) availability is not limiting VO(2) during exercise.The present study evaluated whether intracellular partial pressure of O2 ([Formula: see text]) modulates the muscle O2 uptake (V˙o 2) as exercise intensity increased. Indirect calorimetry followedV˙o 2, whereas nuclear magnetic resonance (NMR) monitored the high-energy phosphate levels, intracellular pH, and intracellular[Formula: see text] in the gastrocnemius muscle of four untrained subjects at rest, during plantar flexion exercise with a constant load at a repetition rate of 0.75, 0.92, and 1.17 Hz, and during postexercise recovery.V˙o 2 increased linearly with exercise intensity and peaked at 1.17 Hz (15.1 ± 0.37 watts), when the subjects could maintain the exercise for only 3 min.V˙o 2 reached a peak value of 13.0 ± 1.59 ml O2 ⋅ min-1 ⋅ 100 ml leg volume-1. The31P spectra indicated that phosphocreatine decreased to 32% of its resting value, whereas intracellular pH decreased linearly with power output, reaching 6.86. Muscle ATP concentration, however, remained constant throughout the exercise protocol. The 1H NMR deoxymyoglobin signal, reflecting the cellular[Formula: see text], decreased in proportion to increments in power output andV˙o 2. At the highest exercise intensity and peakV˙o 2, myoglobin was ∼50% desaturated. These findings, taken together, suggest that the O2 gradient from hemoglobin to the mitochondria can modulate the O2flux to meet the increasedV˙o 2 in exercising muscle, but declining cellular [Formula: see text]during enhanced mitochondrial respiration suggests that O2 availability is not limitingV˙o 2 during exercise.

Myoglobin and hemoglobin rotational diffusion in the cell

The detection of the 1H NMR signal of myoglobin (Mb) in tissue opens an opportunity to examine its cellular diffusion property, which is central to its purported role in facilitating oxygen transport. In perfused myocardium the field-dependent transverse relaxation analysis of the deoxy Mb proximal histidyl NdeltaH indicates that the Mb rotational correlation time in the cell is only approximately 1.4 times longer than it is in solution. Such a mobility is consistent with the theory that Mb facilitates oxygen diffusion from the sarcoplasm to the mitochondria. The microviscosities of the erythrocyte and myocyte environment are different. The hemoglobin (Hb) rotational correlation time is 2.2 longer in the cell than in solution. Because both the overlapping Hb and Mb signals are visible in vivo, a relaxation-based NMR strategy has been developed to discriminate between them.

Metabolic fluctuation during a muscle contraction cycle

Gated31P-nuclear magnetic resonance followed the metabolic fluctuation in rat gastrocnemius muscle during a contraction cycle. Within 16 ms after stimulation, the phosphocreatine (PCr) level drops 11.3% from its reference state. The PCr minimum corresponds closely to the time of maximum force contraction. Pi increases stoichiometrically, while ATP remains constant. During a twitch, PCr hydrolysis produces 3.1 μmol ATP/g tissue, which is substantially higher than the reported 0.3 μmol ATP ⋅ twitch-1 ⋅ g tissue-1 derived from steady-state experiments. The results reveal that a substantial energy fluctuation accompanies a muscle twitch.

Carbon monoxide inhibition of regulatory pathways in myocardium

The 1H nuclear magnetic resonance (NMR) myoglobin (Mb) Val E11 signal provides a unique opportunity to assess the functional role of Mb in the cell. On CO infusion in perfused myocardium, the MbO2 signal at -2.76 parts per million (ppm) gradually disappears, whereas the corresponding MbCO signal emerges at -2.26 ppm, reflecting the state of Mb inhibition. Up to 76.8% MbCO saturation, myocardial O2 consumption (MVO2) remains constant, whereas the rate-pressure product (RPP) has already dropped to 92% of the control level. At 87.6% MbCO saturation, the lactate formation rate has increased by a factor of two, and MVO2 begins to decline. However, the ratio CO/O2 is still 1/10, well below the inhibition threshold for cytochrome oxidase activity. The MVO2 decline in the face of an adequate O2 supply and an unperturbed high-energy phosphate level implies that Mb may play a role in directly regulating respiration, mediated potentially by a shift in NADH/NAD. Although nitrite inhibits Mb, nitrite also directly affects the myocardial function.The 1H nuclear magnetic resonance (NMR) myoglobin (Mb) Val E11 signal provides a unique opportunity to assess the functional role of Mb in the cell. On CO infusion in perfused myocardium, the MbO2 signal at -2.76 parts per million (ppm) gradually disappears, whereas the corresponding MbCO signal emerges at -2.26 ppm, reflecting the state of Mb inhibition. Up to 76.8% MbCO saturation, myocardial O2 consumption (MV˙o 2) remains constant, whereas the rate-pressure product (RPP) has already dropped to 92% of the control level. At 87.6% MbCO saturation, the lactate formation rate has increased by a factor of two, and MV˙o 2 begins to decline. However, the ratio CO/O2 is still 1/10, well below the inhibition threshold for cytochrome oxidase activity. The MV˙o 2 decline in the face of an adequate O2supply and an unperturbed high-energy phosphate level implies that Mb may play a role in directly regulating respiration, mediated potentially by a shift in NADH/NAD. Although nitrite inhibits Mb, nitrite also directly affects the myocardial function.

1H-NMR characterization of the human myocardium myoglobin and erythrocyte hemoglobin signais

Abstract The 1 H-NMR signal of deoxy Mb provides a unique opportunity to measure tissue oxygenation in vivo. To utilize the technique for human application, however, requires a specific spectral characterization of both human Mb and erythrocyte Hb. We report that the proximal histidyl-NH signal of human deoxy Mb resonates at 80.3 ppm at 25°C and maintains a 3.9 ppm separation with the corresponding Hb A signal throughout the physioiogical temperature range. In the particular case of the human thenar muscle, the deoxy Mb signal is clearly detectable without any interference from Hb.

Regulation of respiration in myocardium in the transient and steady state

1H/(31)P NMR has followed the metabolic response to increased work in the glucose- and pyruvate-perfused rat myocardium during a heart cycle and at the steady state. With electrical pacing and dobutamine, the heart O(2) consumption increases by 56%. The phosphocreatine (PCr) level initially declines, but recovers within 15 min to its control level; the oxymyoglobin (MbO(2)) saturation decreases by 15%. Because the MbO(2) signal reflects the intracellular PO(2), the capillary-to-cell O(2) gradient has increased to match the increased O(2) need. However, no transient metabolic fluctuation is observed in either PCr or MbO(2) throughout the entire cardiac cycle in both glucose and pyruvate-/glucose-perfused hearts. No systolic-diastolic variation is detectable under either high workload or hypoxic conditions. The results reveal that neither O(2) nor ADP is regulating respiration under increased energy demand in the steady or transient state.1H/31P NMR has followed the metabolic response to increased work in the glucose- and pyruvate-perfused rat myocardium during a heart cycle and at the steady state. With electrical pacing and dobutamine, the heart O2 consumption increases by 56%. The phosphocreatine (PCr) level initially declines, but recovers within 15 min to its control level; the oxymyoglobin (MbO2) saturation decreases by 15%. Because the MbO2 signal reflects the intracellular [Formula: see text], the capillary-to-cell O2 gradient has increased to match the increased O2 need. However, no transient metabolic fluctuation is observed in either PCr or MbO2 throughout the entire cardiac cycle in both glucose and pyruvate-/glucose-perfused hearts. No systolic-diastolic variation is detectable under either high workload or hypoxic conditions. The results reveal that neither O2 nor ADP is regulating respiration under increased energy demand in the steady or transient state.

Cardioselective dominant-negative thyroid hormone receptor (Δ337T) modulates myocardial metabolism and contractile efficiency

Dominant-negative thyroid hormone receptors (TRs) show elevated expression relative to ligand-binding TRs during cardiac hypertrophy. We tested the hypothesis that overexpression of a dominant-negative TR alters cardiac metabolism and contractile efficiency (CE). We used mice expressing the cardioselective dominant-negative TRbeta(1) mutation Delta337T. Isolated working Delta337T hearts and nontransgenic control (Con) hearts were perfused with (13)C-labeled free fatty acids (FFA), acetoacetate (ACAC), lactate, and glucose at physiological concentrations for 30 min. (13)C NMR spectroscopy and isotopomer analyses were used to determine substrate flux and fractional contributions (Fc) of acetyl-CoA to the citric acid cycle (CAC). Delta337T hearts exhibited rate depression but higher developed pressure and CE, defined as work per oxygen consumption (MVo(2)). Unlabeled substrate Fc from endogenous sources was higher in Delta337T, but ACAC Fc was lower. Fluxes through CAC, lactate, ACAC, and FFA were reduced in Delta337T. CE and Fc differences were reversed by pacing Delta337T to Con rates, accompanied by an increase in FFA Fc. Delta337T hearts lacked the ability to increase MVo(2). Decreases in protein expression for glucose transporter-4 and hexokinase-2 and increases in pyruvate dehydrogenase kinase-2 and -4 suggest that these hearts are unable to increase carbohydrate oxidation in response to stress. These data show that Delta337T alters the metabolic phenotype in murine heart by reducing substrate flux for multiple pathways. Some of these changes are heart rate dependent, indicating that the substrate shift may represent an accommodation to altered contractile protein kinetics, which can be disrupted by pacing stress.

Interaction of Myoglobin with Oleic Acid

Previous studies have shown that palmitate (PA) can interact with myoglobin (Mb). The present study has investigated the interaction of the more soluble unsaturated fatty acid, oleic acid (OA). Indeed, (1)H NMR measurements of the Mb signal during OA titration also show signal changes consistent with specific and non-specific binding. At OA:Mb ratio<4:1, OA perturbs selectively the 8-heme methyl signal, consistent with a local and specific fatty acid-protein interaction. As OA:Mb ratio increases from 4:1 to 40:1, all hyperfine shifted MbCN signals decrease, consistent with a non-selective, global perturbation of the protein. The pH titration analysis indicates that the observed OA methylene signal in the presence of Mb reflects a non-specific interaction and does not originate from a shift in the lamella-micelle equilibrium. Given the OA interaction with Mb, a fatty acid flux model suggests that Mb can play a fatty acid transport role under certain physiological conditions.

1H NMR Approach to Observe Tissue Oxygenation with the Signals of Myoglobin

The myoglobin technique measures oxygen tension in myocytes. It relies on a quantitative measurement of the Val E11 and His F8 signals and an accurate value for the [O2]50 for Mb. Even though the Mb oxygen affinity in the cell is in question, the NMR results still reflect the degree of Mb oxygen saturation. Although magnetic resonance has established a variety of strategies to measure tissue oxygenation, the Mb approach is the most direct and will lead to a better understanding of oxygens role in regulating cellular activity.