The authors have declared that no competing interests exist.
Conceived and designed the experiments: JZ. Performed the experiments: MNJ QH JZ. Analyzed the data: MNJ QH. Contributed reagents/materials/analysis tools: JZ. Wrote the manuscript: MNJ JZ. Oversaw study: JZ.
Decrease of ambient oxygen level has been used in myocytes culture experiments in examining the responsiveness to stress secondary to hypoxia. However, none of these studies measure the myocytes oxygenation levels resulting in ambiguity as to whether there is insufficient oxygen delivery. This study examined the hypothesis that at a basal myocardial work state, adequate myocyte oxygenation would be maintained until extremely low arterial pO2 levels were reached. Myocyte pO2 values in normal dogs were calculated from the myocardial deoxymyoglobin (Mb- δ) levels using 1H-spectroscopy (MRS) and were normalized to Mb-δ obtained after complete LAD occlusion. During Protocol 1 (n = 6), Mb-δ was measured during sequential reductions of the oxygen fraction of inspired gas (FIO2) from 40, 21, 15, 10, and 5%, while in protocol 2 (n = 10) Mb-δ was measured at FIO2 of 3%. Protocol 3 (n = 9) evaluated time course of Mb-δ during prolonged exposure to FIO2 of 5%. Myocardial blood flow (MBF) was measured with microspheres and high energy phosphate (HEP) levels were determined with 31P-MRS. MVO2 progressively increased in response to the progressive reduction of FIO2 that is accompanied by increased LV pressure, heart rate, and MBF. Mb-δ was undetectable during FIO2 values of 21, 15, 10, and 5%. However, FIO2 of 3% or prolonged exposure to FIO2 of 5% caused progressive increases of Mb-δ which were associated with decreases of PCr, ATP and the PCr/ATP ratio, as well as increases of inorganic phosphate. The intracellular PO2 values for 20% reductions of PCr and ATP were approximately 7.4 and 1.9 mmHg, respectively. These data demonstrate that in the in vivo system over a wide range of FIO2 and arterial pO2 levels, the myocyte pO2 values remain well above the Km value with respect to cytochrome oxidase, and oxygen availability does not limit mitochondrial oxidative phosphorylation at 5% FIO2.
In cell culture studies, the different levels of reduction in oxygen in the ambient air are often used as an intervention to examine the hypoxia induced changes of the respective cell types being examined
1H nuclear magnetic resonance spectroscopy can be used to detect myoglobin desaturation. The unpaired electron spin in the heme-Fe(II) complex of deoxymyoglobin (Mb-δ) extends over the proximal histidyl Nδ proton to cause a chemical shift that produces a characteristic resonance on 1H NMR spectroscopy
Consequently, the present study was carried out to examine the effect of decreasing myocyte oxygenation by graded hypoxia on myocardial high energy phosphate content in the intact heart
Studies were performed in 25 adult mongrel dogs of either sex weighing 20–27 kg. All experimental procedures were approved by the University of Minnesota Animal Resources Committee. The investigation conformed to the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health [NIH publication #85–23, revised 1985].
The dogs were anesthetized with sodium pentobarbital (30–35 mg/kg bolus followed by 4 mg/kg/hr, i.v.), intubated and ventilated with a respirator with supplemental oxygen to maintain arterial blood gases within the physiologic range. A heparin-filled polyvinyl chloride catheter, 3.0 mm o.d., was introduced into the right femoral artery and advanced into the ascending aorta. A left thoracotomy was performed through the fourth intercostal space and the heart suspended in a pericardial cradle. A heparin-filled catheter (3.0 mm o.d.) was introduced into the left ventricle through the apical dimple and secured with a purse string suture. A similar catheter was inserted into the left atrium through the atrial appendage. A homemade intra-cardiac vein catheter (0.3 mm o.d) was inserted directly into the great cardiac vein for coronary vein blood sampling. A 1.5–2.0 cm segment of the proximal left anterior descending coronary artery (LAD) was dissected free and a hydraulic occluder constructed of polyvinyl chloride tubing (2.7 mm o.d.) was placed around the artery. A silicone elastomer catheter (0.75 mm i.d.) was placed into the LAD distal to occluder by the method of Gwirtz
Measurements were performed in a 40 cm bore 4.7 Tesla magnet interfaced with a SISCO (Spectroscopy Imaging Systems Corporation, Fremont, CA) console. The left ventricular pressure signal was used to gate MRS data acquisition to the cardiac cycle, while respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions
The method for 1H-MRS detection of the proximal histidyl N-δ proton resonance of Mb-δ has been described in detail
31P MR spectra were acquired in late diastole with a pulse repetition time of 6–7 seconds. This repetition time allowed full relaxation for ATP and Pi resonances, and approximately 90% relaxation for the PCr resonance. PCr resonance intensities were corrected for this minor saturation. RF transmission and signal detection were performed with a 28 mm diameter surface coil. A capillary containing 15 µl of 3M phosphonoacetic acid was placed at the coil center to serve as a reference. The proton signal of the water resonance was used to homogenize the magnetic field and to adjust the position of the animal in the magnet so that the coil was at or near the magnet and gradient isocenter. This was accomplished using a spin-echo experiment with a readout profile. The information gathered in this step was also utilized to determine the spatial coordinates for spectroscopic localization. Chemical shifts were measured relative to PCr which was assigned a chemical shift of −2.55 ppm relative to 85% creatine phosphate at 0 ppm. Spatial localization across the left ventricular wall was performed with the RAPP-ISIS/FSW method. The technical details of this method including voxel profiles, voxel volume, and the accuracy of spatial localization obtained in phantom studies and in vivo have been published elsewhere
Myocardial blood flow was measured using radionuclide labeled microspheres, 15 µm in diameter labeled with 4 different radioisotopes (51Cr, 85Sr, 95Nb and 46Sc). Microsphere suspension containing 2×106 microspheres was injected through the left atrial catheter while a reference sample of arterial blood was withdrawn from the aortic catheter at a rate 15 ml/min beginning 5 seconds before the microsphere injection and continuing for 120 seconds. Radioactivity in the myocardial and blood reference specimens was determined using a gamma spectrometer (Packard Instrument Company, Downers Grove, IL) at window settings chosen for the combination of radioisotopes used during the study. Activity corrected for overlap between isotopes and for background was used to compute blood flow as ml/g of myocardium/minute.
Myocyte oxygenation was estimated from the myoglobin oxygen saturation-PO2 relationship described in equation 1:
In equation 1, Mb-O2 and Mb-δ are fractional contents of oxy-myoglobin and deoxy-myoglobin, respectively, PO2 is the intramyocyte partial pressure of O2, and [PO2]50 is the partial pressure of O2 at which myoglobin is half saturated with O2 (at 37°C this is 2.38 mmHg)
Aortic and left ventricular pressures were measured with fluid filled pressure transducers positioned at mid-chest level and recorded on an 8-channel direct writing recorder (Coulbourne Instrument Company, Lehigh Valley, PA). Left ventricular pressure was recorded at normal and high gain for measurement of end-diastolic pressure. Hemodynamic measurements and 31P and 1H MRS spectra were first obtained under basal conditions.
To examine if more severe hypoxia would produce an association between myoglobin desaturation and reduction of myocardial energetic state and alterations in OXPHOS,
To examine whether the venous O2 would continue to decrease during prolonged hypoxia and whether this would be accompanied by a decrease in myocardial oxygenation level and PCr/ATP,
Hemodynamic data were measured from the chart recordings. 31P spectra were analyzed as described above. Transmural blood flow distribution was determined from the microsphere measurements. Data were analyzed with one-way analysis of variance for repeated measures. A value of p<0.05 was considered significant. When a significant result was found, individual comparisons were made using the method of Sheffé.
The hemodynamic, myocardial blood flow, arterial and coronary vein blood gas and myocardial oxygen consumption, myocardial HEP and oxygenation data are summarized in
Heart Rate, beats/min | Mean Aortic Pressure, mmHg | LV Systolic Pressure, mmHg | LV End-Diastolic Pressure, mmHg | Rate-Pressure Product 1000x (mmHg.beats/min) | |
Baseline | 130±15 | 86±7 | 108±10 | 6±1 | 14.4±2.8 |
21% | 135±17 | 90±8 | 112±13 | 5±1 | 15.7±3.6 |
15% | 136±20 | 87±11 | 111±14 | 6±2 | 15.7±4.0 |
10% | 137±20 | 96±17 | 119±21 | 5±2 | 17.4±5.3 |
5% | 135±16 | 116±17* | 152±22* | 6±2 | 21.5±5.4* |
Occlusion | 127±6 | 86±3 | 104±5* | 6±1 | 14.0±1.0 |
Baseline | 128±6 | 93±4 | 120±6 | 3±1 | 15.4±1.1 |
3% | 124±9 | 128±9* | 168±11* | 7±1* | 21.3±2.6* |
Occlusion | 130±8 | 90±4 | 110±5* | 4±1 | 14.3±1.0 |
Baseline | 132±19 | 103±6 | 124±5 | 4±1 | 16.2±2.2 |
FIO 5% 2mins | 129±6 | 105±7 | 131±12 | 6±1 | 17.0±2.4 |
FIO 5% 4mins | 127±7 | 113±11 | 140±14 | 5±1 | 18.0±2.9 |
FIO 5% 6mins | 125±8 | 120±12 | 146±17 | 7±1 | 18.4±3.0 |
FIO 5% 8mins | 122±6 | 123±10* | 153±12* | 6±1 | 18.8±2.5 |
FIO 5% 10mins | 125±8 | 124±12* | 163±18* | 6±1 | 20.7±3.6* |
FIO 5% 12mins | 125±8 | 131±8* | 175±11* | 7±1 | 21.9±2.2* |
Occlusion | 128±4 | 88±4 | 105±4* | 6±1 | 14.1±1.0 |
Values are means ± SD. * P<0.05 vs Baseline.
Epi | Mid | Endo | End/Epi | Mean Myocardial Blood Flow, ml.min-1.g-1 | AV oxygen Difference, ml 02/100ml | MVO2, ml.min-1.100g-1 | PH-AO | PH-CS | PaO2 (mmHg) | PcsO2 (mmHg) | |
Baseline | 0.46±0.07 | 0.53±0.13 | 0.60±0.16 | 1.27±0.14 | 0.53±0.12 | 11.42±0.71 | 6.37±1.85 | 7.42±0.05 | 7.39±0.03 | 161±31 | 26±2 |
21% | 0.62±0.12 | 0.63±0.18 | 0.68±0.23 | 1.05±0.19 | 0.64±0.17 | 8.01±1.79 | 5.94±2.62 | 7.43±0.05 | 7.40±0.04 | 55±9* | 22±2* |
15% | 0.82±0.08* | 0.91±0.10* | 0.95±0.11* | 1.14±0.12 | 0.89±0.10* | 10.45±0.55 | 9.40±1.13* | 7.51±0.03 | 7.46±0.03 | 53±1* | 19±1* |
10% | 1.21±0.11* | 1.44±0.14* | 1.48±0.09* | 1.20±0.13 | 1.38±0.18* | 8.20±0.88 | 10.11±1.04* | 7.47±0.02 | 7.44±0.02 | 38±4* | 16±2* |
5% | 1.68±0.45* | 1.87±0.56* | 1.71±0.44* | 1.01±0.09 | 1.75±0.48* | 6.70±1.36* | 10.06±2.03* | 7.47±0.03 | 7.46±0.02 | 33±5* | 14±3* |
Occlusion | 0.12±0.01 | 0.05±0.01 | 0.04±0.01 | 0.36±0.07 | 0.07±0.01 | 11.9±0.83 | 0.86±0.17 | 7.40±0.04 | 7.37±0.04 | 101±15 | 22±2 |
Baseline | 0.45± 0.06 | 0.47±0.06 | 0.52±0.07 | 1.16±0.04 | 0.48±0.06 | 11.59±0.42 | 5.49±0.87 | 7.43±0.03 | 7.37±0.03 | 114±11 | 24±1 |
FIO 3% | 1.09±0.16* | 1.21±0.18* | 1.22±0.19* | 1.12±0.04 | 1.17±0.18* | 4.18±0.43* | 5.08±1.24 | 7.52±0.02 | 7.49±0.02 | 21±1* | 12±1* |
Occlusion | 0.10±0.01 | 0.04±0.01 | 0.03±0.01 | 0.35±0.05 | 0.06±0.01 | 11.4±0.70 | 0.74±0.10 | 7.40±0.02 | 7.37±0.05 | 102±14 | 21±2 |
Baseline | 0.57±0.06 | 0.56±0.05 | 0.58±0.04 | 1.05±0.04 | 0.57±0.05 | 10.28±0.72 | 5.92±0.69 | 7.36±0.03 | 7.33±0.02 | 156±19 | 30±2 |
FIO 5% (3mins) | 1.35±0.11* | 1.44±0.18* | 1.43±0.12* | 1.04±0.10 | 1.41±0.12* | 7.81±0.73* | 10.79±1.17* | 7.48±0.04 | 7.47±0.03 | 37±4* | 15±4* |
FIO 5% (8mins) | 2.92±0.26* | 3.30±0.38* | 3.00±0.29* | 1.02±0.04 | 3.07±0.38* | 3.43±0.33* | 8.49±0.84* | 7.46±0.03 | 7.44±0.04 | 21±2* | 11±2* |
Occlusion | 0.10±0.02 | 0.05±0.01 | 0.03±0.01 | 0.35±0.06 | 0.06±0.01 | 11.6±0.83 | 0.80±0.14 | 7.40±0.04 | 7.37±0.05 | 103±12 | 21±3 |
Values are Mean±SD, * p<0.05 vs. Baseline.
Pi/PCr | CP/ATP | CP (Normalized) | ATP (Normalized) | |
BL | ND | 2.34±0.10 | 1.00 | 1.00 |
FIO 21% | ND | 2.37±0.31 | 0.93±0.04 | 0.94±0.04 |
FIO 15% | 0.03±0.03 | 2.33±0.35 | 0.95±0.01 | 0.96±0.07 |
FIO 10% | 0.04±0.03 | 2.25±0.23 | 0.92±0.03 | 0.97±0.03 |
FIO 5% | 0.06±0.03 | 2.09±0.49 | 0.76±0.14 | 0.87±0.08 |
Total Occlusion | 0.80±0.13 | 1.40±0.15† | 0.45±0.04 | 0.77±0.03 |
Baseline | ND | 2.34±0.10 | 1.00 | 1.00 |
FIO 3% | 0.22±0.01 | 1.96±0.12* | 0.73±0.03 | 0.84±0.02 |
Total occluded | 0.78±0.10 | 1.35±0.10† | 0.40±0.04 | 0.75±0.02 |
Baseline | ND | 2.28±0.06 | 1.00 | 1.00 |
FIO 5% 4mins after | 0.04±0.02 | 2.20±0.06 | 0.92±0.01 | 0.97±0.01 |
FIO 5% 8mins after | 0.21±0.01 | 1.91±0.11* | 0.77±0.05 | 0.91±0.02 |
FIO 5% 10mins after | 0.33±0.04 | 1.83±0.13* | 0.69±0.07 | 0.86±0.04 |
FIO 5% 14mins after | 0.54±0.05 | 1.71±0.19* | 0.62±0.08 | 0.82±0.05 |
Total occlusion | 0.76±0.12 | 1.38±0.12† | 0.42±0.04 | 0.76±0.02 |
Values are means ± SE. * P<0.05 vs. Baseline. † P<0.01 vs. Baseline.
Deoxymyoglobin | |
BL | 0 |
FIO 21% | 0 |
FIO 15% | 0 |
FIO 10% | 0 |
FIO 5% | 0 |
Total Occlusion | 1 |
Baseline | 0 |
FIO 3% (2 min) | 0.21±0.04* |
FIO 3% (3.5 min) | 0.26±0.03* |
FIO 3% (5 min) | 0.33±0.06* |
FIO 3% (6.5 min) | 0.45±0.07* |
FIO 3% (8 min) | 0.57±0.04* |
Total occluded | 1 |
Baseline | 0 |
FIO 5% (2 min) | 0 |
FIO 5% (3.5 min) | 0 |
FIO 5% (5 min) | 0 |
FIO 5% (6.5 min) | 0 |
FIO 5% (8 min) | 0.26±0.06* |
FIO 5% (10 min) | 0.29±0.06* |
FIO 5% (14 min) | 0.39±0.07* |
FIO 5% (16 min) | 0.50±0.05* |
Total occluded | 1 |
Values are means ± SE. * P<0.05 vs. Baseline.
Hemodynamic measurements during each experimental condition are shown in
DMB = level of deoxymyoglobin normalized to total LAD occlusion; MBF = myocardial blood flow rate (ml per minute per gram myocardium measured by microspheres at each experimental conditions). *, p<0.01 VS Baseline; #, p<0.01 VS. LAD occlusion.
To examine whether more severe hypoxia would produce an association between myoglobin de-saturation and reduction of myocardial HEP, 10 dogs were exposed to 3% FIO2. The mean aortic pressure and LV systolic pressure increased substantially resulting in a significant increase in rate pressure product [
Protocol 3 was performed to examine whether prolonged 5% FIO2 would result in continuous reduction of coronary venous pO2 and therefore detectable Mb-δ. Prolonged 5% FIO2 caused a significant increase in LV systolic pressure and subsequent RPP. MVO2 increased as a result of increase in MBF but there was a significant decrease in HEP which was associated with an increase in deoxymyoglobin after 8 minutes exposure. It was evident that when the 5% FIO2 was maintained the venous pO2 continued to decrease. Once it was below 14 mm Hg, the Mb-δ became detectable.
Decreasing FIO2 beyond 5% or prolonged exposure to low FIO2 was associated with a decrease in PCr/ATP and increase in Pi/PCr which was associated with a decrease in intracellular PO2. The relationships of PCr and ATP with intracellular PO2 are shown in
The present study examined whether mitochondria lack oxygen under the extreme low FIO2 conditions, and if so, how long it takes the compensation systems to fail. We examined whether the 1H- Mb-d and 31P- MR spectroscopic measurements can detect the signals of reduction of oxygen delivery to the mitochondria. Prior studies did not measure Mb-d, therefore, no evidence of decrease of oxygen delivery to the mitochondria under these conditions. The findings from the present study demonstrate that the in vivo compensate mechanisms can maintain the normal mtOXPHOS even during extreme low FIO2. The present study has further elucidated the response of the myocardial oxidative phorsphorylation (OXPHOS) regulation to hypoxia. Firstly, it has reiterated the hemodynamic response of the in vivo heart to graded hypoxia as a result of a decrease in the fractional inspired oxygen concentration. Secondly, it has depicted the increase in transmural blood flow during hypoxia, which helps in maintaining a normal mitochondrial OXPHOS. Thirdly, it has defined the relationships between myocyte PO2 and HEP levels in vivo myocardium under conditions where myocardial flow is not limited.
During myocardial hypoxia, the lactate production and glycolysis pathway of ATP production are increased
Our study shows that the mean arterial pressure, heart rate, left ventricular systolic and end diastolic pressure and rate pressure product are maintained with graded reductions in FIO2 up to 10% [
The graded hypoxia caused a proportional increase in myocardial blood flow up to 5% FIO2 [
Under normal baseline conditions, the myocardium has adequate supply of oxygen for oxidative phosphorylation and oxygen does not seem to play a regulatory role. Even at high cardiac work states, no deoxymyoglobin signal is detected despite a decrease in high energy phosphates
The higher PO2 required for decrease in HEP in the present study suggests that there were other factors which are affecting the intracellular PO2 in the graded coronary artery stenoses setting. Indeed, the response of myocardial HEP levels to decreased coronary perfusion can be affected by changes in myocardial oxygen demand. Gregg initially described that myocardial oxygen consumption can be influenced by coronary flow and perfusion pressures. Several mechanisms have been proposed to modulate oxygen demand during hypoperfusion. The pressure in the coronary system may distend the ventricles, leading to increased contractility and oxygen consumption by an increase in sarcomere length or ventricular stiffness
Another important question to address is why any oxygen limitation occurs at an intramyocyte PO2 that far exceeds the apparent Michaelis-Menten constant of cytochrome oxidase with respect to O2. It was shown that the critical PO2 at which oxidative phosphorylation began to decrease in isolated mitochondria was approximately 0.01 mmHg, which is more than 100 fold lower than the PO2 at which HEP levels started to decrease in the present study. This could be attributed to inhomogeneities of O2 availability at the cellular level, if the PO2 gradient between the cytosol and the inner mitochondrial membrane was significant. However, mathematical models of O2 diffusion suggest very low perimitochondrial O2 gradient
In conclusion, during graded hypoxia caused by decreased FIO2, significant loss of PCr occurred at an intracellular PO2 of 7.4 mmHg. Thus, in normal in vivo heart, oxygen availability plays an important role in regulation of oxidative phosphorylation only when mean intracellular PO2 falls below 7.4 mmHg.