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Role of Oxygen in Limiting Respiration in theIn SituMyocardium
J Mol Cell Cardiol 30, 2651–2655 (1998) Article No. mc980823
Role of Oxygen in Limiting Respiration in the In Situ Myocardium Ulrike Kreutzer, Yousry Mekhamer, Tuan-Khanh Tran and Thomas Jue Department of Biological Chemistry Department, University of California Davis, Davis, CA 94616-8635, USA (Received 26 May 1998, accepted in revised form 10 September 1998) U. K, Y. M, T.-K. T T. J. Role of Oxygen in Limiting Respiration in the In Situ Myocardium. Journal of Molecular and Cellular Cardiology (1998) 30, 2651–2655. 1H NMR has now detected the proximal histidyl NdH myoglobin (Mb) signal from the myocardium in situ. Upon ligation of the left anterior descending coronary artery in the rat myocardium, the deoxy Mb signal appears at 78 ppm During dopamine infusion at up to 80 lg/kg/min, the heart rate pressure product (RPP) increases by a factor of 2, the phosphocreatine (PCr) decreases by 17%, and the ratio of the change in inorganic phosphate over PCr (DPi/PCr) increases by 0.2. However, no deoxy myoglobin signal is detected. Oxygen availability does not appear to limit oxygen consumption nor oxidative phosphorylation under dopamine enhanced work state in myocardium. 1998 Academic Press
K W: Myoglobin; NMR; Respiration; Myocardium; Oxidative phosphorylation.
Introduction In muscle tissue, oxygen consumption can vary across a wide range to meet the energy demand. How the cell regulates its oxygen need is dependent upon a co-ordinated set of controls along the oxygen cascade from air to mitochondria. Although researchers have defined various physiological controls, the key regulatory step remains elusive. Some have posited that respiration is controlled by the cytochrome aa3 oxidation state (Hoppeler and Lindstedt, 1985; Stainsby, 1989). Others have contended that O2 diffusion or flow is the limiting step (Gregg, 1963; Roca et al., 1989; Hogan et al., 1993). The debate over the regulatory mechanism would clarify substantially if the intracellular oxygen level were known; at least the oxygen availability under different physiological states would signal whether the key step is upstream in the vasculature or downstream in the mitochondria dependent reactions. Unfortunately, measuring intracellular oxygen under physiological conditions has posed
formidable experimental challenges (Whalen, 1971; Woods et al., 1989; Parsons et al., 1990; Holland et al., 1993). Recently, a 1H NMR technique has emerged to monitor the intracellular oxygenation with the proximal histidy NdH and the Val E11 signals of myoglobin (Mb) (Kreutzer et al., 1992). Perfused heart experiments have established that such an approach can quantify the relationship between cellular P2 and bioenergetics and define the critical P2 in normal and post-ischemic myocardium (Kreutzer and Jue, 1995; Chung and Jue, 1996). The results suggest that oxygen is not a limiting factor. However, the buffer perfused heart is a simplified model, which does not respond to intricate vascular controls and does not engender sufficient confidence in the respiratory regulation in vivo. Moreover, whether the Mb technique is still applicable in myocardium in situ is still an open question. Indeed, the Mb signal is detectable in the in situ myocardium of an open-chest, instrumented rat heart. As oxygen consumption and the rate pressure
Please address all correspondence to: Dr Thomas Jue, Med: Biological Chemistry, University of California Davis, Davis, CA 956168635, USA
0022–2828/98/122651+05 $30.00/0
1998 Academic Press
2652
U. Kreutzer et al.
product (RPP) increase with dopamine infusion, no deoxy Mb appears. However, when RPP has increased by a factor of 2, PCr has decreased by 17% (Zhang et al., 1995). Although cellular oxygen level is unperturbed, the PCr decline already indicates a limitation in oxidative phosphorylation. These results demonstrate that the Mb technique can detect intracellular P2 in the in situ myocardium, and suggest that oxygen is not limiting respiration under enhanced workstates.
Materials and Methods Whole animal preparation Male Sprague–Dawley rats (450g) were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital. A Nellcor pulse oximeter monitored the arterial oxygen saturation and pulse rate via the tail artery. The left jugular vein and the right common carotid artery were cannulated with polyethylene tubing. A catheter in the left ventricle monitored the left ventricular pressure and heart rate. Anesthesia was maintained by intravenous infusion pentobarbital at 15 mg/kg/h. A tracheotomy was performed, and a rodent respirator ventilated the animal with room air. Stroke volume was maintained at 3 ml; ventilation rate was 65 strokes/min; arterial oxygen saturation was above 90%. A medial sternotomy exposed the heart. The pericardium was removed, and a suture (silk 6-0) was placed around the left anterior descending coronary artery (LAD) about 2 mm below its origin. The animal was placed on a lucite holder, and a concentric 31P/1H surface coil was placed over the heart. After a control period of 30–45 min, dopamine was infused at a continuous rate of 80 lg/kg/min for a period of 30 min. The infusion was stopped, and the animal was allowed to recover for 30 min. the suture around the LAD was ligated during the last phase of the experiment.
NMR methods Spectra were recorded with a GE Omega 7T 15cm-diameter horizontal bore spectrometer system. A concentric 31P/1H coil system was utilized. The 1 H and 31P 90° pulse at the coil was 25 and 35 ls, respectively.
A modified DANTE sequence was utilized to suppress the water signal and excite the Mb resonances (Morris and Freeman, 1978). The sequence was tailored to give a maximum excitation at 76 ppm. A typical 1H spectrum consisted of 10 000 transients, requiring a total acquisition time of 7 min. The spectral width was 60 kHz; data block size was 4k. All spectra were referenced to water at 4.76 ppm, at 25°C, which in turn was calibrated against DSS. Repetition time was 40 ms, and transmitter power was set to give a 90° pulse at the coil center. For the 31P spectra, the spectral width was 6 kHz; data block size was 1k. Repetition time was 1s. Total number of scans was 128. 1 H and 31P spectra were acquired during the control period, during dopamine infusion, and during LAD ligation. Data are reported as means ±... (n=4). Statistical significance was determined by Student’s t-test with P<0.05.
Results Figure 1 shows the 1H spectra from the myocardium of an open-chest rat (n=4). During the control period, no signal is detected in the region between 100–60 ppm [Fig. 1A(a)]. With the infusion of dopamine at 80 lg/kg/min, still no signal appears [Fig. 1A(b)]. However, upon ligation of the LAD, the deoxy Mb proximal histidyl NdH emerges at 78 p.p.m. [Fig. 1A(c)]. Given the deoxy Mb signal to noise ratio of approximately 20:1, which is assumed to reflect the fully deoxygenated state, a 10% change in Mb desaturation would be detectable. On the upfield shoulder at 75 p.p.m. is a hint of a second signal that is assigned to the b deoxy Hb proximal histidyl NdH signal. The corresponding 31P signals are shown in Figure 1B. During the control period, the signals of PCr, ATP, and Pi are clearly visible [Fig. 1B(a)]. However, during dopamine infusion, the PCr decreases and the Pi increases [Fig. 1B(b)]. Upon ligation of the LAD, the PCr signal disappears, and the Pi signal increases [Fig. 1B(c)]. In Figure 2, the physiological traces indicate that the dopamine infusion increases the left ventricular systolic pressure by 60%. The end diastolic pressure does not change substantially. The heart rate also increases by 25% upon dopamine infusion. As a consequence, the RPP increases by 100% during dopamine infusion. The dose–response curve indicates that RPP rises with dopamine concentration and reaches a maximal steady state level at 80 lg/ kg/min infusion.
Oxygen and Oxidative Phosphorylation in Myocardium
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Figure 1 1H and 31spectra from in situ myocardium. A. (a) During the control period, no 1H signal appears in the 100–60 ppm region. (b) Upon infusion with 80 lg/kg/min of dopamine, the spectral region shows no signal of the proximal histidyl N8H of Mb. (c) After LAD ligation, the deoxy Mb signal appears at 78 ppm. The upfield shoulder corresponds to the proximal histidyl N8H signal of the b-subunit of Hb. B. 31P spectra from in situ myocardium (a) during control (b) during 80 lg/kg/min of dopamine, and (c) after LAD ligation. The PCr signal decreases and the Pi signal increases during control and dopamine infusion. When the ATP and PCr levels are normalized to 100% and DPi/PCr to 0% during control, dopamine infusion produces a significant PCr drop to 82.7±3.9% of control. DPi/PCr increases to 0.24±0.08 of control. ATP does not show any significant change. Values are means±.. (n=4). Significant difference between control and dopamine infused hearts is based on paired t-test (P<0.05).
Discussion 1
H NMR can detect the Mb signal in the in situ myocardium (Chen et al., 1997) [Fig. 1]. In contrast to the global ischemia in the perfused heart experiments, ligating the LAD affects only a local region. Still, the proximal histidyl NdH signal of deoxy Mb is clearly detectable. Unlike skeletal muscle, blood-perfused myocardium may be poised at the brink of oxygen limitation and may contain partially-saturated Mb, consistent with its role in facilitating oxygen diffusion (Wittenberg, 1970). The results from Figure 1Aa show Mb is not partially saturated under physiological conditions, and raise the question about its cellular role. No deoxy Mb signal is detected under control or with dopamine
infusion. If the deoxy Mb signal during LAD ligation is presumed to be 100% desaturated Mb, then the resting MbO2 in the myocardium is clearly well saturated. Along with the Mb signal at 78 ppm there is a hint of an upfield shoulder at 75 ppm. Such a peak is consistent with the resonance of the proximal histidyl NdH signals from the b subunit of Hb (Kreutzer et al., 1993). The shoulder peak does not correspond to a sub-population of Mb, which would require a temperature gradient of almost 13°C. Observing only the b proximal histidyl NdH is consistent with the higher oxygen affinity of the Hb a subunit (Viggiano et al., 1979). Upon dopamine infusion, RPP increases by a factor of 2, the PCr decreases by 17%, and the DPi/
2654
U. Kreutzer et al.
in oxidative phosphorylation, which is potentially related to oxygen/carbon precursor delivery to the mitochondria, and not to oxygen availability. The Mb technique to measure intracellular P2 has then gleaned some provocative observations and will continue with further experiments to shed light on the control of oxidative phosphorylation in the myocardium in vivo.
Acknowledgement We gratefully acknowledge the funding from the NIH GM 57355 and scientific discussion with Dr. Youngran Chung.
References Figure 2 Physiological response during dopamine infusion experiment: (A) LVSP (filled symbols) and heart rate (open symbols) increase as dopamine infusion (hatched bar) begins. LVSP increases by 60%, whereas the heart rate increases by 25%. (B) Upon dopamine infusion, RPP increases by a factor 2 from its basal state. With LAD ligation, RPP drops to 40% of control. Under control condition, the heart rate was 228±13 beats/ min, left ventricular systolic pressure was between 88±13 mmHg, and RPP was 20.1±1.7×103mmHg/ min.
PCr ratio increases to 0.24. These observations are consistent with previous canine studies (Zhang et al., 1995). If the change in RPP is associated with a corresponding increase in MV2, then any limitation in oxygen availability should correspond with an increased Mb desaturation. No deoxy Mb signal, however, is detected. Whether Mb desaturates at V2max is still unclear, since V2 of dopamine stimulated heart is not at V2max. From the perfused heart studies, Mb desaturation should precede any changes in PCr. When cellular P2 declines to a critical level, between 2–4 mm Hg, PCr level has already fallen significantly (Kreutzer et al., 1992; Kreutzer and Jue, 1995; Chung and Jue, 1996). In contrast, the in situ myocardium spectra show no deoxy Mb signal, despite a drop in PCr. Cellular P2 limitation does not give rise to the DPi/PCr ratio. Neither convective O2 delivery or capillary O2 conductance appears limiting under these experimental conditions. An ample supply of oxygen in face of a DPi/PCr increase, if the ratio reflects ischemic onset, would imply a limitation
C W, Z J, E MH, Z Y, Z XH, W C, C Y, M H, U K, 1997. Determination of deoxymyoglobin changes during graded myocardial ischemia: an in vivo 1H HMR spectroscopy study. Magn Reson Med 38: 193–197. C Y, J T, 1996. Cellular response to reperfused oxygen in postischemic myocardium. Am J Physiol 271: H1166–H1173. G DE, 1963. The effect of coronary perfusion pressure and coronary flow on oxygen usage of the myocardium. Circulation 13: 497–500. H MC, B DE, W PD, 1993. Effect of blood flow reduction on maximal O2 uptake in canine gastrocnemius muscle in situ. J Appl Physiol 74: 1742– 1747. H SK, K RP, S MM, D’A MJ, G JC, 1993. Imaging oxygen tension in liver and spleen by 19F NMR. Magn Reson Med 29: 446–458. H H, L SL, 1985. Malleability of skeletal muscle in overcoming limitations: structural elements. J Exp Biol 115: 355–364. K U, J T, 1995. Critical intracellular oxygen in the myocardium as determined with the 1H NMR signal of myoglobin. Am J Physiol 268: H1675–H1681. K U, W DS, J T, 1992. Observing the 1H NMR signal of myoglobin val e11 in myocardium: an index of cellular oxygenation. Proc Natl Acad Sci USA 89: 4731–4733. K U, C Y, B D, J T, 1993. 1H NMR characterization of the human myocardium myoglobin and erythrocyte hemoglobin signals. Biochim Biophys Acta 1161: 33–37. M GA, F R, 1978. Selective excitation in fourier transform nuclear magnetic resonance. J Magn Reson 29: 433–462. P WJ, R JC, B RP, G JC, P CA, 1990. Dynamic mechanisms of cardiac oxygenation during brief ischemia and reperfusion. Am J Physiol 259: H1477–H1485. R J, H MC, S D, D DE, H P, G-
Oxygen and Oxidative Phosphorylation in Myocardium R, U O, W PD, 1989. Evidence for tissue diffusion limitation of VO2 max in normal humans. J Appl Physiol 67: 291–299. S WN, 1989. Oxidation/reduction state of cytochrome oxidase during repetitive contractions. J Appl Physiol 67: 2158–2162. V G, H NT, H C, 1979. Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide. Biochemistry 18: 5238–5247. W WJ, 1971. Intracellular pO2 in heart and skeletal muscle. Physiologist 14: 69–82.
2655
W JB, 1970. Myoglobin-facilitated oxygen diffusion: role of myoglobin in oxygen entry into muscle. Physiol Rev 50: 559–636. W RK, D JW, G JF, M PD, S HM, 1989. Spectral spatial ESR imaging as a method of noninvasive biological oximetry. J Magn Reson 85: 50–59. Z J, D DJ, X Y, Z Y, P G, M H, H K, F AHL, B RJ, U K, 1995. Transmural bioenergetic responses of normal myocardium to high workstates. Am J Physiol 268: H1891–H1905.
Role of Oxygen in Limiting Respiration in the In Situ Myocardium Ulrike Kreutzer, Yousry Mekhamer, Tuan-Khanh Tran and Thomas Jue Department of Biological Chemistry Department, University of California Davis, Davis, CA 94616-8635, USA (Received 26 May 1998, accepted in revised form 10 September 1998) U. K, Y. M, T.-K. T T. J. Role of Oxygen in Limiting Respiration in the In Situ Myocardium. Journal of Molecular and Cellular Cardiology (1998) 30, 2651–2655. 1H NMR has now detected the proximal histidyl NdH myoglobin (Mb) signal from the myocardium in situ. Upon ligation of the left anterior descending coronary artery in the rat myocardium, the deoxy Mb signal appears at 78 ppm During dopamine infusion at up to 80 lg/kg/min, the heart rate pressure product (RPP) increases by a factor of 2, the phosphocreatine (PCr) decreases by 17%, and the ratio of the change in inorganic phosphate over PCr (DPi/PCr) increases by 0.2. However, no deoxy myoglobin signal is detected. Oxygen availability does not appear to limit oxygen consumption nor oxidative phosphorylation under dopamine enhanced work state in myocardium. 1998 Academic Press
K W: Myoglobin; NMR; Respiration; Myocardium; Oxidative phosphorylation.
Introduction In muscle tissue, oxygen consumption can vary across a wide range to meet the energy demand. How the cell regulates its oxygen need is dependent upon a co-ordinated set of controls along the oxygen cascade from air to mitochondria. Although researchers have defined various physiological controls, the key regulatory step remains elusive. Some have posited that respiration is controlled by the cytochrome aa3 oxidation state (Hoppeler and Lindstedt, 1985; Stainsby, 1989). Others have contended that O2 diffusion or flow is the limiting step (Gregg, 1963; Roca et al., 1989; Hogan et al., 1993). The debate over the regulatory mechanism would clarify substantially if the intracellular oxygen level were known; at least the oxygen availability under different physiological states would signal whether the key step is upstream in the vasculature or downstream in the mitochondria dependent reactions. Unfortunately, measuring intracellular oxygen under physiological conditions has posed
formidable experimental challenges (Whalen, 1971; Woods et al., 1989; Parsons et al., 1990; Holland et al., 1993). Recently, a 1H NMR technique has emerged to monitor the intracellular oxygenation with the proximal histidy NdH and the Val E11 signals of myoglobin (Mb) (Kreutzer et al., 1992). Perfused heart experiments have established that such an approach can quantify the relationship between cellular P2 and bioenergetics and define the critical P2 in normal and post-ischemic myocardium (Kreutzer and Jue, 1995; Chung and Jue, 1996). The results suggest that oxygen is not a limiting factor. However, the buffer perfused heart is a simplified model, which does not respond to intricate vascular controls and does not engender sufficient confidence in the respiratory regulation in vivo. Moreover, whether the Mb technique is still applicable in myocardium in situ is still an open question. Indeed, the Mb signal is detectable in the in situ myocardium of an open-chest, instrumented rat heart. As oxygen consumption and the rate pressure
Please address all correspondence to: Dr Thomas Jue, Med: Biological Chemistry, University of California Davis, Davis, CA 956168635, USA
0022–2828/98/122651+05 $30.00/0
1998 Academic Press
2652
U. Kreutzer et al.
product (RPP) increase with dopamine infusion, no deoxy Mb appears. However, when RPP has increased by a factor of 2, PCr has decreased by 17% (Zhang et al., 1995). Although cellular oxygen level is unperturbed, the PCr decline already indicates a limitation in oxidative phosphorylation. These results demonstrate that the Mb technique can detect intracellular P2 in the in situ myocardium, and suggest that oxygen is not limiting respiration under enhanced workstates.
Materials and Methods Whole animal preparation Male Sprague–Dawley rats (450g) were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital. A Nellcor pulse oximeter monitored the arterial oxygen saturation and pulse rate via the tail artery. The left jugular vein and the right common carotid artery were cannulated with polyethylene tubing. A catheter in the left ventricle monitored the left ventricular pressure and heart rate. Anesthesia was maintained by intravenous infusion pentobarbital at 15 mg/kg/h. A tracheotomy was performed, and a rodent respirator ventilated the animal with room air. Stroke volume was maintained at 3 ml; ventilation rate was 65 strokes/min; arterial oxygen saturation was above 90%. A medial sternotomy exposed the heart. The pericardium was removed, and a suture (silk 6-0) was placed around the left anterior descending coronary artery (LAD) about 2 mm below its origin. The animal was placed on a lucite holder, and a concentric 31P/1H surface coil was placed over the heart. After a control period of 30–45 min, dopamine was infused at a continuous rate of 80 lg/kg/min for a period of 30 min. The infusion was stopped, and the animal was allowed to recover for 30 min. the suture around the LAD was ligated during the last phase of the experiment.
NMR methods Spectra were recorded with a GE Omega 7T 15cm-diameter horizontal bore spectrometer system. A concentric 31P/1H coil system was utilized. The 1 H and 31P 90° pulse at the coil was 25 and 35 ls, respectively.
A modified DANTE sequence was utilized to suppress the water signal and excite the Mb resonances (Morris and Freeman, 1978). The sequence was tailored to give a maximum excitation at 76 ppm. A typical 1H spectrum consisted of 10 000 transients, requiring a total acquisition time of 7 min. The spectral width was 60 kHz; data block size was 4k. All spectra were referenced to water at 4.76 ppm, at 25°C, which in turn was calibrated against DSS. Repetition time was 40 ms, and transmitter power was set to give a 90° pulse at the coil center. For the 31P spectra, the spectral width was 6 kHz; data block size was 1k. Repetition time was 1s. Total number of scans was 128. 1 H and 31P spectra were acquired during the control period, during dopamine infusion, and during LAD ligation. Data are reported as means ±... (n=4). Statistical significance was determined by Student’s t-test with P<0.05.
Results Figure 1 shows the 1H spectra from the myocardium of an open-chest rat (n=4). During the control period, no signal is detected in the region between 100–60 ppm [Fig. 1A(a)]. With the infusion of dopamine at 80 lg/kg/min, still no signal appears [Fig. 1A(b)]. However, upon ligation of the LAD, the deoxy Mb proximal histidyl NdH emerges at 78 p.p.m. [Fig. 1A(c)]. Given the deoxy Mb signal to noise ratio of approximately 20:1, which is assumed to reflect the fully deoxygenated state, a 10% change in Mb desaturation would be detectable. On the upfield shoulder at 75 p.p.m. is a hint of a second signal that is assigned to the b deoxy Hb proximal histidyl NdH signal. The corresponding 31P signals are shown in Figure 1B. During the control period, the signals of PCr, ATP, and Pi are clearly visible [Fig. 1B(a)]. However, during dopamine infusion, the PCr decreases and the Pi increases [Fig. 1B(b)]. Upon ligation of the LAD, the PCr signal disappears, and the Pi signal increases [Fig. 1B(c)]. In Figure 2, the physiological traces indicate that the dopamine infusion increases the left ventricular systolic pressure by 60%. The end diastolic pressure does not change substantially. The heart rate also increases by 25% upon dopamine infusion. As a consequence, the RPP increases by 100% during dopamine infusion. The dose–response curve indicates that RPP rises with dopamine concentration and reaches a maximal steady state level at 80 lg/ kg/min infusion.
Oxygen and Oxidative Phosphorylation in Myocardium
2653
Figure 1 1H and 31spectra from in situ myocardium. A. (a) During the control period, no 1H signal appears in the 100–60 ppm region. (b) Upon infusion with 80 lg/kg/min of dopamine, the spectral region shows no signal of the proximal histidyl N8H of Mb. (c) After LAD ligation, the deoxy Mb signal appears at 78 ppm. The upfield shoulder corresponds to the proximal histidyl N8H signal of the b-subunit of Hb. B. 31P spectra from in situ myocardium (a) during control (b) during 80 lg/kg/min of dopamine, and (c) after LAD ligation. The PCr signal decreases and the Pi signal increases during control and dopamine infusion. When the ATP and PCr levels are normalized to 100% and DPi/PCr to 0% during control, dopamine infusion produces a significant PCr drop to 82.7±3.9% of control. DPi/PCr increases to 0.24±0.08 of control. ATP does not show any significant change. Values are means±.. (n=4). Significant difference between control and dopamine infused hearts is based on paired t-test (P<0.05).
Discussion 1
H NMR can detect the Mb signal in the in situ myocardium (Chen et al., 1997) [Fig. 1]. In contrast to the global ischemia in the perfused heart experiments, ligating the LAD affects only a local region. Still, the proximal histidyl NdH signal of deoxy Mb is clearly detectable. Unlike skeletal muscle, blood-perfused myocardium may be poised at the brink of oxygen limitation and may contain partially-saturated Mb, consistent with its role in facilitating oxygen diffusion (Wittenberg, 1970). The results from Figure 1Aa show Mb is not partially saturated under physiological conditions, and raise the question about its cellular role. No deoxy Mb signal is detected under control or with dopamine
infusion. If the deoxy Mb signal during LAD ligation is presumed to be 100% desaturated Mb, then the resting MbO2 in the myocardium is clearly well saturated. Along with the Mb signal at 78 ppm there is a hint of an upfield shoulder at 75 ppm. Such a peak is consistent with the resonance of the proximal histidyl NdH signals from the b subunit of Hb (Kreutzer et al., 1993). The shoulder peak does not correspond to a sub-population of Mb, which would require a temperature gradient of almost 13°C. Observing only the b proximal histidyl NdH is consistent with the higher oxygen affinity of the Hb a subunit (Viggiano et al., 1979). Upon dopamine infusion, RPP increases by a factor of 2, the PCr decreases by 17%, and the DPi/
2654
U. Kreutzer et al.
in oxidative phosphorylation, which is potentially related to oxygen/carbon precursor delivery to the mitochondria, and not to oxygen availability. The Mb technique to measure intracellular P2 has then gleaned some provocative observations and will continue with further experiments to shed light on the control of oxidative phosphorylation in the myocardium in vivo.
Acknowledgement We gratefully acknowledge the funding from the NIH GM 57355 and scientific discussion with Dr. Youngran Chung.
References Figure 2 Physiological response during dopamine infusion experiment: (A) LVSP (filled symbols) and heart rate (open symbols) increase as dopamine infusion (hatched bar) begins. LVSP increases by 60%, whereas the heart rate increases by 25%. (B) Upon dopamine infusion, RPP increases by a factor 2 from its basal state. With LAD ligation, RPP drops to 40% of control. Under control condition, the heart rate was 228±13 beats/ min, left ventricular systolic pressure was between 88±13 mmHg, and RPP was 20.1±1.7×103mmHg/ min.
PCr ratio increases to 0.24. These observations are consistent with previous canine studies (Zhang et al., 1995). If the change in RPP is associated with a corresponding increase in MV2, then any limitation in oxygen availability should correspond with an increased Mb desaturation. No deoxy Mb signal, however, is detected. Whether Mb desaturates at V2max is still unclear, since V2 of dopamine stimulated heart is not at V2max. From the perfused heart studies, Mb desaturation should precede any changes in PCr. When cellular P2 declines to a critical level, between 2–4 mm Hg, PCr level has already fallen significantly (Kreutzer et al., 1992; Kreutzer and Jue, 1995; Chung and Jue, 1996). In contrast, the in situ myocardium spectra show no deoxy Mb signal, despite a drop in PCr. Cellular P2 limitation does not give rise to the DPi/PCr ratio. Neither convective O2 delivery or capillary O2 conductance appears limiting under these experimental conditions. An ample supply of oxygen in face of a DPi/PCr increase, if the ratio reflects ischemic onset, would imply a limitation
C W, Z J, E MH, Z Y, Z XH, W C, C Y, M H, U K, 1997. Determination of deoxymyoglobin changes during graded myocardial ischemia: an in vivo 1H HMR spectroscopy study. Magn Reson Med 38: 193–197. C Y, J T, 1996. Cellular response to reperfused oxygen in postischemic myocardium. Am J Physiol 271: H1166–H1173. G DE, 1963. The effect of coronary perfusion pressure and coronary flow on oxygen usage of the myocardium. Circulation 13: 497–500. H MC, B DE, W PD, 1993. Effect of blood flow reduction on maximal O2 uptake in canine gastrocnemius muscle in situ. J Appl Physiol 74: 1742– 1747. H SK, K RP, S MM, D’A MJ, G JC, 1993. Imaging oxygen tension in liver and spleen by 19F NMR. Magn Reson Med 29: 446–458. H H, L SL, 1985. Malleability of skeletal muscle in overcoming limitations: structural elements. J Exp Biol 115: 355–364. K U, J T, 1995. Critical intracellular oxygen in the myocardium as determined with the 1H NMR signal of myoglobin. Am J Physiol 268: H1675–H1681. K U, W DS, J T, 1992. Observing the 1H NMR signal of myoglobin val e11 in myocardium: an index of cellular oxygenation. Proc Natl Acad Sci USA 89: 4731–4733. K U, C Y, B D, J T, 1993. 1H NMR characterization of the human myocardium myoglobin and erythrocyte hemoglobin signals. Biochim Biophys Acta 1161: 33–37. M GA, F R, 1978. Selective excitation in fourier transform nuclear magnetic resonance. J Magn Reson 29: 433–462. P WJ, R JC, B RP, G JC, P CA, 1990. Dynamic mechanisms of cardiac oxygenation during brief ischemia and reperfusion. Am J Physiol 259: H1477–H1485. R J, H MC, S D, D DE, H P, G-
Oxygen and Oxidative Phosphorylation in Myocardium R, U O, W PD, 1989. Evidence for tissue diffusion limitation of VO2 max in normal humans. J Appl Physiol 67: 291–299. S WN, 1989. Oxidation/reduction state of cytochrome oxidase during repetitive contractions. J Appl Physiol 67: 2158–2162. V G, H NT, H C, 1979. Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide. Biochemistry 18: 5238–5247. W WJ, 1971. Intracellular pO2 in heart and skeletal muscle. Physiologist 14: 69–82.
2655
W JB, 1970. Myoglobin-facilitated oxygen diffusion: role of myoglobin in oxygen entry into muscle. Physiol Rev 50: 559–636. W RK, D JW, G JF, M PD, S HM, 1989. Spectral spatial ESR imaging as a method of noninvasive biological oximetry. J Magn Reson 85: 50–59. Z J, D DJ, X Y, Z Y, P G, M H, H K, F AHL, B RJ, U K, 1995. Transmural bioenergetic responses of normal myocardium to high workstates. Am J Physiol 268: H1891–H1905.