Effect of fatty acid interaction on myoglobin oxygen affinity and triglyceride metabolism

Recent studies have suggested myoglobin (Mb) may have other cellular functions in addition to storing and transporting O2. Indeed, NMR experiments have shown that the saturated fatty acid (FA) palmitate (PA) can interact with myoglobin (Mb) in its ligated state (MbCO and MbCN) but does not interact with Mb in its deoxygenated state. The observation has led to the hypothesis that Mb can also serve as a fatty acid transporter. The present study further investigates fatty acid interaction with the physiological states of Mb using the more soluble but unsaturated fatty acid, oleic acid (OA). OA binds to MbCO but does not bind to deoxy Mb. OA binding to Mb, however, does not alter its O2 affinity. Without any Mb, muscle has a significantly lower level of triglyceride (TG). In Mb knock-out (MbKO) mice, both heart and skeletal muscles have lower level of TG relative to the control mice. Training further decreases the relative TG in the MbKO skeletal muscle. Nevertheless, the absence of Mb and lower TG level in muscle does not impair the MbKO mouse performance as evidenced by voluntary wheel running measurements. The results support the hypothesis of a complex physiological role for Mb, especially with respect to fatty acid metabolism

Effect of fatty acid interaction on myoglobin oxygen affinity and triglyceride metabolism.

Recent studies have suggested myoglobin (Mb) may have other cellular functions in addition to storing and transporting O2. Indeed, NMR experiments have shown that the saturated fatty acid (FA) palmitate (PA) can interact with myoglobin (Mb) in its ligated state (MbCO and MbCN) but does not interact with Mb in its deoxygenated state. The observation has led to the hypothesis that Mb can also serve as a fatty acid transporter. The present study further investigates fatty acid interaction with the physiological states of Mb using the more soluble but unsaturated fatty acid, oleic acid (OA). OA binds to MbCO but does not bind to deoxy Mb. OA binding to Mb, however, does not alter its O2 affinity. Without any Mb, muscle has a significantly lower level of triglyceride (TG). In Mb knock-out (MbKO) mice, both heart and skeletal muscles have lower level of TG relative to the control mice. Training further decreases the relative TG in the MbKO skeletal muscle. Nevertheless, the absence of Mb and lower TG level in muscle does not impair the MbKO mouse performance as evidenced by voluntary wheel running measurements. The results support the hypothesis of a complex physiological role for Mb, especially with respect to fatty acid metabolism.

Imaging apolipoprotein AI in vivo.

Coronary disease risk increases inversely with high-density lipoprotein (HDL) level. The measurement of the biodistribution and clearance of HDL in vivo, however, has posed a technical challenge. This study presents an approach to the development of a lipoprotein MRI agent by linking gadolinium methanethiosulfonate (Gd[MTS-ADO3A]) to a selective cysteine mutation in position 55 of apo AI, the major protein of HDL. The contrast agent targets both liver and kidney, the sites of HDL catabolism, whereas the standard MRI contrast agent, gadolinium-diethylenetriaminepentaacetic acid-bismethylamide (GdDTPA-BMA, gadodiamide), enhances only the kidney image. Using a modified apolipoprotein AI to create an HDL contrast agent provides a new approach to investigate HDL biodistribution, metabolism and regulation in vivo.

'It's hollow': the function of pores within myoglobin.

Despite a century of research, the cellular function of myoglobin (Mb), the mechanism regulating oxygen (O(2)) transport in the cell and the structure-function relationship of Mb remain incompletely understood. In particular, the presence and function of pores within Mb have attracted much recent attention. These pores can bind to Xe as well as to other ligands. Indeed, recent cryogenic X-ray crystallographic studies using novel techniques have captured snapshots of carbon monoxide (CO) migrating through these pores. The observed movement of the CO molecule from the heme iron site to the internal cavities and the associated structural changes of the amino acid residues around the cavities confirm the integral role of the pores in forming a ligand migration pathway from the protein surface to the heme. These observations resolve a long-standing controversy - but how these pores affect the physiological function of Mb poses a striking question at the frontier of biology.

Oximetry with the NMR signals of hemoglobin Val E11 and Tyr C7.

The NMR visibility of the signals from erythrocyte hemoglobin (Hb) presents an opportunity to assess the vascular PO(2) (partial pressure of oxygen) in vivo to gather insight into the regulation of O(2) transport, especially in contracting muscle tissue. Some concerns, however, have arisen about the validity of using the Val E11 signal as an indicator of PO(2), since its intensity depends on tertiary structural changes, in contrast to the quaternary structure changes associated with relaxed (R) and tense (T) transition during O(2) binding. We have examined the Val E11 and Tyr C7 signal intensity as a function of Hb saturation by developing an oximetry system, which permits the comparative analysis of the NMR and spectrophotometric measurements. The spectrophotometric assay defines the Hb saturation level at a given PO(2) and yields standard oxygen-binding curves. Under defined PO(2) and Hb saturation values, the NMR measurements have determined that the Val E11 signal, as well as the Tyr C7 signal, tracks closely Hb saturation and can therefore serve as a vascular oxygen biomarker.

Interaction of fatty acid with myoglobin.

Upon titration with palmitate, the (1)H NMR spectra of metmyoglobin cyanide (MbCN) reveal a selective perturbation of the 8 heme methyl, consistent with a specific interaction of myoglobin (Mb) with fatty acid. Other detectable hyperfine shifted resonances of the heme group remain unchanged. Mb also enhances fatty acid solubility, as reflected in a more intense methylene peak of palmitate in Mb solution than in Tris buffer. Ligand binding analysis indicates an apparent palmitate dissociation constant (K(d)) of 43microM. These results suggest that Mb can bind fatty acid and may have a role in facilitating fatty acid transport in the cell.

Blood flow and metabolic regulation in seal muscle during apnea.

In order to examine myoglobin (Mb) function and metabolic responses of seal muscle during progressive ischemia and hypoxemia, Mb saturation and high-energy phosphate levels were monitored with NMR spectroscopy during sleep apnea in elephant seals (Mirounga angustirostris). Muscle blood flow (MBF) was measured with laser-Doppler flowmetry (LDF). During six, spontaneous, 8-12 min apneas of an unrestrained juvenile seal, apneic MBF decreased to 46+/-10% of the mean eupneic MBF. By the end of apnea, MBF reached 31+/-8% of the eupneic value. The t(1/2) for 90% decline in apneic MBF was 1.9+/-1.2 min. The initial post-apneic peak in MBF occurred within 0.20+/-0.04 min after the start of eupnea. NMR measurements revealed that Mb desaturated rapidly from its eupenic resting level to a lower steady state value within 4 min after the onset of apnea at rates between 1.7+/-1.0 and 3.8+/-1.5% min(-1), which corresponded to a muscle O(2) depletion rate of 1-2.3 ml O(2) kg(-1) min(-1). High-energy phosphate levels did not change with apnea. During the transition from apnea to eupnea, Mb resaturated to 95% of its resting level within the first minute. Despite the high Mb concentration in seal muscle, experiments detected Mb diffusing with a translational diffusion coefficient of 4.5 x 10(-7) cm(2) s(-1), consistent with the value observed in rat myocardium. Equipoise P(O(2)) analysis revealed that Mb is the predominant intracellular O(2) transporter in elephant seals during eupnea and apnea.

Determination of myoglobin concentration in blood-perfused tissue.

The standard method for determining the myoglobin (Mb) concentration in blood-perfused tissue often relies on a simple but clever differencing algorithm of the optical spectra, as proposed by Reynafarje. However, the underlying assumptions of the differencing algorithm do not always lead to an accurate assessment of Mb concentration in blood-perfused tissue. Consequently, the erroneous data becloud the understanding of Mb function and oxygen transport in the cell. The present study has examined the Mb concentration in buffer and blood-perfused mouse heart. In buffer-perfused heart containing no hemoglobin (Hb), the optical differencing method yields a tissue Mb concentration of 0.26 mM. In blood-perfused tissue, the method leads to an overestimation of Mb. However, using the distinct (1)H NMR signals of MbCO and HbCO yields a Mb concentration of 0.26 mM in both buffer- and blood-perfused myocardium. Given the NMR and optical data, a computer simulation analysis has identified some error sources in the optical differencing algorithm and has suggested a simple modification that can improve the Mb determination. Even though the present study has determined a higher Mb concentration than previously reported, it does not alter significantly the equipoise PO(2), the PO(2) where Mb and O(2) contribute equally to the O(2) flux. It also suggests that any Mb increase with exercise training does not necessarily enhance the intracellular O(2) delivery.

Anisotropy and temperature dependence of myoglobin translational diffusion in myocardium: implication for oxygen transport and cellular architecture.

Pulsed field gradient NMR methods have determined the temperature-dependent diffusion of myoglobin (Mb) in perfused rat myocardium. Mb diffuses with an averaged translational diffusion coefficient (DMb) of 4.24-8.37x10(-7)cm2/s from 22 degrees C to 40 degrees C and shows no orientation preference over a root mean-square displacement of 2.5-3.5 microm. The DMb agrees with the value predicted by rotational diffusion measurements. Based on the DMb, the equipoise diffusion PO2, the PO2 in which Mb-facilitated and free O2 diffusion contribute equally to the O2 flux, varies from 2.72 to 0.15 in myocardium and from 7.27 to 4.24 mmHg in skeletal muscle. Given the basal PO2 of approximately 10 mmHg, the Mb contribution to O2 transport appears insignificant in myocardium. In skeletal muscle, Mb-facilitated diffusion begins to contribute significantly only when the PO2 approaches the P50. In marine mammals, the high Mb concentration confers a predominant role for Mb in intracellular O2 transport under all physiological conditions. The Q10 of the DMb ranges from 1.3 to 1.6. The Mb diffusion data indicate that the postulated gel network in the cell must have a minimum percolation cutoff size exceeding 17.5 A and does not impose tortuosity within the diffusion root mean-square displacement. Moreover, the similar Q10 for the DMb of solution versus cell Mb suggests that any temperature-dependent alteration of the postulated cell matrix does not significantly affect protein mobility.

Myoglobin translational diffusion in rat myocardium and its implication on intracellular oxygen transport.

Current theory of respiratory control invokes a role of myoglobin (Mb)-facilitated O2 diffusion in regulating the intracellular O2 flux, provided Mb diffusion can compete effectively with free O2 diffusion. Pulsed-field gradient NMR methods have now followed gradient-dependent changes in the distinct 1H NMR gamma CH3 Val E11 signal of MbO2 in perfused rat myocardium to obtain the endogenous Mb translational diffusion coefficient (D(Mb)) of 4.24 x 10(-7) cm2 s(-1) at 22 degrees C. The D(Mb) matches precisely the value predicted by in vivo NMR rotational diffusion measurements of Mb and shows no orientation preference. Given values in the literature for the Krogh's free O2 diffusion coefficient (K0), myocardial Mb concentration and a partial pressure of O2 that half saturates Mb (P50), the analysis yields an equipoise diffusion P(O2) of 1.77 mmHg, where Mb and free O2 contribute equally to the O2 flux. In the myocardium, Mb-facilitated O2 diffusion contributes increasingly more than free O2 diffusion when the P(O2) falls below 1.77 mmHg. In skeletal muscle, the P(O2) must fall below 5.72 mmHg. Altering the Mb P50 induces modest change. Mb-facilitated diffusion has a higher poise in skeletal muscle than in myocardium. Because the basal P(O2) hovers around 10 mmHg, Mb does not have a predominant role in facilitating O2 transport in myocardium but contributes significantly only when cellular oxygen falls below the equipoise diffusion P(O2).

Investigation of bioactive NO-scavenging role of myoglobin in myocardium.

Because nitric oxide (NO) can react with myoglobin (Mb) to oxidize the heme Fe(II) to Fe(III), the appearance of metmyoglobin (metMb) during bradykinin stimulation underpins the hypothesis that Mb acts as an NO scavenger in the cell. Although some experiments have detected the reporter metMb signal in the -3.7 ppm spectral region, others have not corroborated the finding. Because metMb also has characteristic hyperfine-shifted signals in the 40-100 ppm spectral region, detection of these signals would confirm the presence of metMb. Perfused rat myocardium study has examined this spectral region in a range of bradykinin infusion protocols. Although bradykinin elicits a set of physiological responses, consistent with the induction of NO, the (1)H nuclear magnetic resonance spectra in all experiments reveal no detectable metMb signals. Moreover, in the perfused myocardium model, the bradykinin-induced decline in myocardial oxygen consumption does not appear to arise only from NO binding to cytochrome oxidase.

Role of myoglobin as a scavenger of cellular NO in myocardium.

Recent studies have detected a (1)H nuclear magnetic resonance (NMR) reporter signal of metmyoglobin (metMb) during bradykinin stimulation of an isolated mouse heart. The observation has led to the hypothesis that Mb reacts with cellular nitric oxide (NO). However, the hypothesis depends on an unequivocal detection of metMb signals in vivo. In solution, nitrite oxidization of Mb produces a characteristic set of paramagnetically shifted (1)H NMR signals. In the upfield spectral region, MbO(2) and MbCO exhibit the gammaCH(3) Val E11 signals at -2.8 and -2.4 ppm, respectively. In the same spectral region, nitrite oxidation of Mb produces a set of signals at -3.7 and -4.7 ppm at 35 degrees C. Previous studies have confirmed the visibility of metMb signals in perfused rat myocardium. With bradykinin infusion, perfusion pressure and rate-pressure product decrease, consistent with endogenous NO formation. However, neither myocardial O(2) consumption nor high-energy phosphate levels, as reflected in the (31)P NMR signals, show any significant change. Bradykinin still triggers a similar physiological response even in the presence of CO that is sufficient to inhibit 86% Mb. In all cases, the (1)H NMR spectra from perfused rat myocardium reveal no metMb signals. The results suggest that bradykinin-induced NO does not interact significantly with cellular Mb to produce an NMR-detectable quantity of metMb in the perfused rat myocardium. As a consequence, the experiments cannot confirm the intriguing proposal that Mb acts as a cellular NO scavenger.

Detection of myoglobin desaturation in Mirounga angustirostris during apnea.

1H NMR solution-state study of elephant seal (Mirounga angustirostris) myoglobin (Mb) and hemoglobin (Hb) establishes the temperature-dependent chemical shifts of the proximal histidyl N(delta)H signal, which reflects the respective intracellular and vascular PO2 in vivo. Both proteins exist predominantly in one major isoform and do not exhibit any conformational heterogeneity. The Mb and Hb signals are detectable in M. angustirostris tissue in vivo. During eupnea M. angustirostris muscle maintains a well-saturated MbO2. However, during apnea, the deoxymyoglobin proximal histidyl N(delta)H signal becomes visible, reflecting a declining tissue PO2. The study establishes a firm methodological basis for using NMR to investigate the metabolic responses during sleep apnea of the elephant seal and to secure insights into oxygen regulation in diving mammals.