Regulation of oxygen diffusion in hypoxic isolated cardiac myocytes

In the normal beating heart, oxygen pressure (PO2) gradients between capillary blood and intracellular space are so large that cytosolic PO2 may decline to around PO2 at half saturation of myoglobin (2-5 Torr). Hence, a decrease in capillary blood PO2 of a few Torr would easily deplete oxygen in mitochondria if PO2 gradients are unchanged. The aim of the present study was to demonstrate, in a single isolated cardiac myocyte of the rat, a mechanism that reduces PO2 gradients in hypoxia so that oxygenation of the intracellular space would be sustained. Using a newly developed microspectrophotometric device, we were able to follow changes in cytosolic PO2 of an individual ventricular myocyte in an hypoxic medium. For extracellular PO2 of 4.4 Torr, we found an elevation (2.1 Torr) of the cytosolic PO2 when oxygen consumption of the cell was abolished by 2 mM NaCN, thus demonstrating PO2 gradients from extracellular medium to cytosolic space in a single individual cardiomyocyte. The magnitude of these PO2 gradients was reduced as extracellular PO2 was further lowered, and they were no longer detectable for extracellular PO2 of 0.6 Torr. To further elucidate physiological effects of the PO2-dependent changes in PO2 gradients demonstrated above, we conducted a simulation of ischemia in a single cardiac myocyte. The stop-flow procedure (simulated ischemia) quickly decreased cytosolic PO2 from 7.3 to 1.8 Torr in 5 min, while the rate of fall of PO2 considerably decreased when the cytosolic PO2 decreased to < 2 Torr. Consequently, even 30 min after the onset of the stop flow, cytosolic PO2 was significantly higher than that of the anoxic perfusion. These results together suggest that in severe hypoxia oxygenation of the intracellular space might be partially maintained by relative elevation of cytosolic PO2, resulting from progressive decrease in PO2 gradients from extracellular space to cytosol.

Determination of PO2 and its heterogeneity in single capillaries

We have applied the phosphorescence lifetime technique (Vanderkooi, J. M., G. Maniara, T. J. Green, and D. F. Wilson. J. Biol. Chem. 262: 5476-5482, 1987) to determine oxygen tension in single capillaries of the hamster retractor muscle. Palladium meso-tetra(4-carboxyphenyl)porphine (10 mg/ml, pH 7.40, bound to bovine serum albumin) was used as the phosphorescent oxygen sensor. Our measurement system consisted of a microscope configured for epi-illumination, a strobe flash lamp, a 430-nm bandpass excitation filter, and a 630-nm cut-on emission filter. A rectangular diaphragm was used to limit the illumination field to 10 microns x 10 microns, and an end-window photomultiplier tube was used to detect the phosphorescence signal, which was then input to an analog-to-digital board in a personal computer. In vitro calibrations were carried out at 37 degrees C on samples flowing through a glass capillary tube (diameter, 300 microns) at four different O2 concentrations (0, 2.5, 5, and 7.5%). In vivo tests were carried out on arterioles, capillaries, and venules of the retractor muscle of anesthetized hamsters. The phosphorescent compound was administered by injection into a jugular vein (20 mg/kg). Phosphorescence decay curves were analyzed by a new model of heterogeneous oxygen distribution in the excitation/emission volume. Mean Po2 values and the local Po2 gradients within the excitation/ emission volume were calculated from phosphorescence life-times obtained from individual decay curves. The time course of Po2 obtained during 0.5-s measurement periods (5 decay curves at 0.1-s intervals) at a given site along a capillary indicated the presence of a gradient in Po2 within the plasma space between and near red blood cells. Similar Po2 gradients were also detected in arterioles and venules. Mean Po2 values for arterioles, capillaries, and venules over the 0.5-s observation period were 27 +/- 5, 14 +/- 2, and 11 +/- 3 (SD) mmHg, respectively. The magnitude of the Po2 gradient in the arterioles, capillaries, and venules was 6 +/- 1, 4 +/- 1, and 2 +/- 1 mmHg/micron, respectively.

Calculated intra- and extracellular PO2 gradients in heavily working red muscle

A recently introduced three-dimensional analytical model of O2 diffusion to heavily working muscle that considers myoglobin-facilitated O2 diffusion inside the muscle fiber and a carrier-free layer separating erythrocytes and fiber is able to furnish the following new insights in O2 supply to red muscle at high performance. 1) Fiber PO2 profiles are essentially flat, and the major PO2 gradients are located in the perierythrocytic region, in good agreement with experimental findings [T. E. J. Gayeski and C. R. Honig, Am. J. Physiol. 251 (Heart Circ. Physiol. 20): H789-H799, 1986]. No specialized anatomical pericapillary barrier structure is required to explain these results. 2) A functional barrier to O2 diffusion has been identified that consists of the carrier-free layer and of the pericapillary muscle fiber portions. There are three reasons that make these structures act as a diffusion barrier: a “geometric reason,” a “diffusivity-related reason,” and a “myoglobin-related reason.” 3) PO2 fields of adjacent red blood cells (RBCs) practically do not interact. 4) Small scale heterogeneities in capillary and RBC spacing are compensated for by high myoglobin-facilitated fiber diffusivity. Limiting factor for diffusional O2 transport is the number of RBCs present on the fiber surface.