A 31P-NMR study of tissue respiration in working muscle during reduced 0, delivery conditions MICHAEL

C. HOGAN,

SHOKO

NIOKA,

WILLIAM

F. BRECHUE,

AND

BRITTON

dog

CHANCE

Division

of Physiology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623; Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and Department of Pharmacology, College of Medicine, University of Florida,

Gainesville, Florida 32610 HOGAN,MICHAELC.,SHOKONIOKA,WILLIAM F. BRECHUE, the mitochondrial AND BRITTONCHANCE.A 31P-NMR study of tissue respiration tors of respiration

in working dog muscle during reduced 0, delivery conditions. J. Appl. Physiol. 73(4): 166%1670,1992.-To investigate the role of tissue oxygenation as one of the control factors regulating tissue respiration, 31P-nuclear magnetic resonance spectroscopy (31P-NMR) was used to estimate muscle metabolites in isolated working muscle during varied tissue oxygenation conditions. 0, delivery (muscle blood flow X arterial 0, content) was varied to isolated in situ working dog gastrocnemius (n = 6) by decreases in arterial PO, (hypoxemia; H) and by decreases in muscle blood flow (ischemia; I). 0, uptake (VO,) was measured at rest and during work at two or three stimulation intensities (isometric twitch contractions at 3, 5, and occasionally 7 Hz) during three separate conditions: normal 0, delivery (C) and reduced 0, delivery during H and I, with blood flow controlled by pump perfusion. Biochemical metabolites were measured during the last 2 min of each 3-min work period by use of 31PNMR, and arterial and venous blood samples were drawn and muscle blood flow measured during the last 30 s of each work period. Muscle [ATP] did not fall below resting values at any work intensity, even during O,-limited highly fatiguing work, and was never different among the three conditions. Muscle 0, delivery and 00, were significantly less (P < 0.05) at the highest work intensities for both I and H than for C but were not different between H and I. As TO, increased with stimulation intensity, a larger change in any of the proposed regulators of tissue respiration (ADP, Pi, ATP/ADP Pi, and phosphocreatine) was required during H and I than during C to elicit a given vo2, but requirements were similar for H and I. These results suggest that 1) the sensitivity of mitochondrial respiration to the proposed regulators of tissue respiration can be altered by the level of tissue oxygenation and 2) under the conditions of this study, the bioenergetics of H and I were similar. l

fatigue; skeletal muscle; gas exchange; respiration; phosphocreatine

exercise; mitochondrial

RECENT RESULTS(12), collected using standard biochem-

ical methods, demonstrated that the factors believed to regulate tissue respiration during work in an intact muscle (such as ADP, Pi, and phosphocreatine) may be modulated by an interaction with tissue oxygenation levels. It was found that, depending on the degree of tissue oxygenation, quite different amounts of any of these proposed regulators of tissue respiration were required to attain similar levels of 0, uptake (VO&, suggesting that 1662

0161-7567/92

$2.00 Copyright

sensitivity to these proposed regulacould be altered by tissue oxygenation levels. Although the results of this prior study (12) are in agreement with others (l&17,20,31) showing that alterations in 0, availability have an effect on cellular metabolism at similar submaximal VO,, they (12) indicated also that tissue oxygenation levels may be a factor in the control of tissue respiration. This finding supports the hypothesis that there can be alterations in the proposed regulators of tissue respiration as mitochondrial PO, is reduced, as a compensatory mechanism to maintain vo, (4,31). The mitochondrial PO, necessary to maintain high rates of respiration is not yet clearly established (9). It was originally suggested as a result of work on isolated mitochondria that maximal rates of respiration could be maintained at very low levels of O,, inasmuch as the “true” K, for 0, (PO, at half-maximal respiration) is ~0.03-0.1 Torr (5), so that 0, control of respiration was considered to be important only at low rate-limiting levels. However, it was suggested at. that time that K, was not constant but proportional to VO,, as set by regulators of mitochondrial metabolism. In intact tissues, it appears that the “apparent” K, for 0, is variable and to some extent may shift, depending on the metabolic rate and mitochondrial PO,. Wilson and colleagues demonstrated in isolated mitochondria (32), cells without myoglobin (26), and recently in cardiac myocytes (28) that perhaps there is not simply a minimal “critical” value for [0,] within the cell below which oxidative phosphorylation becomes compromised but, rather, a range of 0, values that influences both metabolic and respiratory states of the cell. In addition, Jones and colleagues also demonstrated that the Co,] required for half-maximal oxidation of cytochrome c was significantly lower in isolated mitochondria than in isolated liver cells (18) and was doubled (from 4 to 8 Torr) in isolated working compared with isolated resting myocytes (19). Jones et al. (18, 19) proposed that even small 0, gradients have an essential role in determining the apparent K, of cells and tissues; such effects have also been important in the studies of Wilson and colleagues (26,28,32). Thus vo2 appears to alter K, and established 0, gradients. It is believed that these differences between the true and apparent K, for 0, and the critical PO, as measured in intact cells and isolated mitochondria may be due to diffusion limitations (28)

0 1992 the American

Physiological

Society

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MUSCLE

RESPIRATORY

REGULATION

and mitochondrial heterogeneity (18). These studies suggest a wider role for tissue PO, as a factor in the control of tissue respiration. It has also been reported recently (29) that the mechanical responses of contracting muscle to ischemic and hypoxemic hypoxia may not be the same and that the reduction in VO, during ischemia, in contrast to hypoxemia, may not be a result of 0, lack in the mitochondria. If in fact hypoxic hypoxia reduces tissue respiration through mechanisms different from ischemic hypoxia, then it might be expected that the metabolic and bioenergetic changes within the cell should be different at similar vo2 for these two conditions. The purpose of the current study was to use 3fP-nuclear magnetic resonance spectroscopy (31P-NMR) to measure theemetabolic state of working muscle when 0, delivery (Qo,; arterial 0, content X muscle blood flow) was reduced by equal amounts during hypoxemia and ischemia to further investigate 1) the role of tissue oxygenation in the regulation of tissue respiration and 2) whether the bioenergetics of hypoxemia and ischemia are different at similar QO,. METHODS

Six adult mongrel dogs of either sex with a weight range of 15-25 kg were anesthetized with pentobarbital sodium (30 mg/kg) and given maintenance doses as required. The dogs were intubated with cuffed endotracheal tubes. Heating pads were used to maintain esophageal temperature at -37OC. The animals were given heparin (1,500 U/kg) after the surgery. Ventilation was maintained with a ventilator (model 613, Harvard) at a rate that maintained the PCO, at -35 Torr. Surgical preparation. The left gastrocnemius-flexor digitorum superficialis muscle complex (for convenience referred to as gastrocnemius) was isolated, as described previously (15). Briefly, a medial incision was made through the skin of the left hindlimb from the midthigh to the ankle. The sartorius, gracilis, semitendinosus, and semimembranosus muscles, which overlie the gastrocnemius, were doubly ligated and cut between the ties. All vessels draining into the popliteal vein except those from the gastrocnemius were ligated to isolate the venous outflow from the gastrocnemius. The arterial circulation to the gastrocnemius was isolated by ligating all vessels from the femoral and popliteal artery that did not enter the gastrocnemius. The left popliteal vein was cannulated, and the venous outflow from the isolated muscle was returned to the animal via a jugular catheter. The right femoral artery was catheterized for arterial blood sampling. This catheter was connected to the left femoral artery so that the isolated muscle was perfused by blood from this contralateral artery. Perfusion was accomplished either directly from the contralateral by systemic pressure (self perfused) or via a Sigmamotor pump to control flow. A pressure transducer in this line at the head of the muscle constantly monitored perfusion pressure. A carotid artery was also catheterized to monitor systemic blood pressure. The left sciatic nerve, which innervates the gastrocnemius, was doubly ligated and cut between ties. To prevent cooling and drying, all exposed

AS

STUDIED

BY

31P-NMR

1663

tissues were covered with saline-soaked gauze and a sheet of Saran-Wrap. After the muscle was surgically isolated, the Achilles tendon was attached to an isometric myograph (model FTlO, Grass Instruments) that was fixed to the magnet to measure tension development. The hindlimb was fixed at the knee and ankle and attached to rods that positioned the muscle in the center of the magnet. Weights were used at the end of each experiment to calibrate the tension myograph. Isometric muscle contractions (twitch) were elicited by stimulation of the sciatic nerve with square wave impulses (4-6 V) of 0.2 ms. The muscle was stimulated at 3 contractions/s for 3 min and then at 5 contractions/s for 3 min. If there was little fatigue at this work intensity, then the stimulation intensity was increased to 7 contractions/s for another 3 min. A stimulation rate of 5 contractions/s has been shown to achieve the maximal Tjoz for this in situ muscle model for twitch contractions (30), and although this cannot be directly compared with in vivo conditions, it represents the highest VO, that can be obtained under these conditions and is especially useful in comparing different treatments. Before each contraction period, the resting muscle was passively stretched until the tension development during a twitch contraction was maximal. Experimental protocol. After the surgery, the animal was placed within the magnet so that the exposed muscle was centered. Before the first contraction period, the blood supply to the isolated muscle was switched from self-perfused to pump perfused, and enough time was allowed for conditions to stabilize at a blood flow similar to the self-perfused level. Each experiment (n = 6) consisted of three separate contraction periods for the isolated muscle. Each contraction period, consisting of 3 min at 3 Hz, 3 min at 5 Hz, and if necessary 3 min at 7 Hz, was separated from the others by 20-30 min of rest. These three contraction periods were 1) the control condition (C), consisting of normal arterial PO, (PaoJ and normal muscle blood flow maintained by keeping muscle perfusion pressure at 140-150 mmHg; 2) the ischemic condition (I), consisting of normal Paoz but with muscle blood flow reduced by keeping the muscle perfusion pressure at ~100 mmHg; and 3) the hypoxemic condition (H), consisting of reducing the fraction of inspired 0, that the dog breathed to produce a low Pap, (27 t 1 Torr) and keeping muscle perfusion pressure similar to C. An attempt was made during each experiment to match the Qo, values between the H and I conditions at each stimulation intensity. The desired condition was initiated - 10 min before the start of the contraction period (to allow equilibration during H), and the order of the conditions was varied so that all six possible orders of the three conditions were each conducted once. Measurements. Arterial blood samples from the carotid artery and venous samples from the left popliteal vein as close to the gastrocnemius as possible were drawn anaerobically at the end of each rest period and during the last 20 s of each of the two stimulation periods and were kept on ice. Barbee et al. (2), using muscle contractions similar to those used in this investigation, determined that a near-steady-state flow and VO, had been achieved by the end of 2 min. Venous blood flow measure-

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1664

MUSCLE

RESPIRATORY

REGULATION

ments were made at the same time the blood samples were drawn by timed blood collections into a graduated cylinder. Fatigue was measured as the percent decline in developed tension from the maximal developed tension during each 3-min stimulation period and is reported as a ratio of final developed tension during a stimulation period to the maximal developed tension during that stimulation period. Blood PO,, Pco,, and pH were measured with a blood gas analyzer (model ABLl, Radiometer) at 37OC while hemoglobin concentration, percent 0, saturation, percent CO saturation, and 0, content were measured with an IL 282 CO-oximeter. These instruments were calibrated before and often throughout each experiment. The concentrations of ATP, phosphocreatine (PCr), and inorganic phosphate (Pi) were determined from 31PNMR spectra obtained with a 2.1-T 30-cm-bore superconducting magnet (Otsuka Electronics USA). A 2.5cm single-turn double-tuned copper coil located on the gastrocnemius muscle transmitted a radio-frequency pulse (46.94 MHz, 20- to 30-ps width for a m90° pulse) and received a phosphorus magnetic resonance from the muscle for 80 ms. Pulses taken every 4 s were accumulated to improve the signal-to-noise ratio. Free induction delays were typically collected in 2-min scans (the last 2 min of each 3-min work period). Shimming was obtained to get a narrow water peak (usually a 0.2-ppm-half-line width) in proton magnetic resonance spectroscopy. Saturation factors for ATP, PCr, and Pi were obtained by calculating the ratios of fully relaxed spectra areas acquired at interpulse intervals of 20 s to the areas obtained at interpulse intervals of 4 s. The data were Fourier transformed and phased. The areas of six peaks (P-ATP, wATP, T-ATP, PCr, Pi, and phosphomonoester) were measured by area analysis program (American Innovision). PCr and Pi were quantitated from the ratios of the PCr and Pi peak areas to the NMR area of P-ATP. The [ATP] (3.5 mM) obtained from the previous biochemical assay (12) in this isolated muscle was used as an internal calibration source. Saturation factors for PCr and Pi were 1.14 and 1.07. Intracellular [H+] was calculated in four of the animals (data were considered too noisy for this calculation in the other 2 animals) from the chemical shift of Pi (22, 23). The values of [PCr] calculated in this muscle at rest are similar to those obtained from biochemical measurements in TABLE 1. Arterial

blood hemodynamic

AS STUDIED

BY

this muscle previously [ 13.5 mM (12) and 13.2 mM (25)], and our values of [PC,] and [Pi] at rest are similar to the values of 12-13 and 3.9 mM, respectively, measured by Connett et al. (7, 8) in dog gracilis muscle at rest. ADP was calculated from the creatine kinase equilibrium (8, 22) by use of the muscle [H+] and the measured values of total [Cr] (12), [PCr], and [ATP]. During the maximal stimulation rate for each condition, a Bohr integration technique was used to calculate the muscle 0, diffusing capacity (Dm,J that could account for the fall in the measured PO, and 0, content from artery to vein by use of a simple model of capillary gas exchange based on diffusion, as outlined previously (13). From the fall in capillary PO, calculated with this value of Dmoz, a muscle mean capillary PO, (Pco2) was calculated. The Fick principle was used to calculate muscle Vo2. Statistics. Three-way analysis of variance was used for the statistical analysis. Duncan’s multiple range test was used to determine where differences occurred. Standard linear correlation tests were run on several variables to determine their relationship. In all statistical analyses, P = 0.05. RESULTS Mean weight of the gastrocnemius muscles (n = 6) removed after the end of the experiment was 73 t 3 (SE) g. During H, there was substantial fatigue (a fall in developed tension ~10%) at 5 Hz in all six muscles, whereas, during I, one of the six muscles had to be driven to 7 Hz to produce fatigue, and finally, during C, five of the muscles had to be worked at 7 Hz before fatigue set in. The principal variables of 0, transport and acid-base balance in the blood perfusing the muscles during the three conditions (C, H, and I) for rest and the two stimulation intensities (3 Hz and the maximal stimulation intensity) are presented in Table 1. Paoz was significantly less for H at each of the three collection periods, as was expected with the lower fraction of inspired 0, breathed in that condition. This lower Pa,, resulted in lower 0, hemoglobin saturation values and thereby significantly reduced arterial 0, contents. The other variables reflecting O,-carrying capacity and the acid-base status of the blood perfusing the muscles were quite similar for the three conditions and did not change from rest to work. Table 2 presents the data relevant to 0, transport, uti-

and acid-base status during three conditions Stimulation

Pa,-, PacoP, PH, [Hb], BP,,

Torr Torr g/100 ml mmHg

27+1* 36+4 7.40+0.04 12.2k0.4 118+7

79+3

78+9

33+4 7.37kO.02 12.7kO.4 100~18

33+3 7.4OkO.03 12.7kO.3 113+8

26&l* 38+3 7.36kO.04 12.21k0.4 145215

Values are means + SE; IZ = 6 (except pH,, for which n = 4). H, hypoxemia, arterial pH; [Hb], hemoglobin concn; BP,, muscle arterial blood pressure. intensity.

31P-NMR

74+3 33+3 7.37kO.02 12.720.4 90+7* I, ischemia, * Significantly

75+2 32k4 7.39kO.03 12.720.4 135klO C, control. different

at rest and at two work intensities Intensity

75+2 33k2 7.36kO.02 12.5kO.4 97*6*

41-t4 7.36kO.03 12.220.4 15Ok13 Paoz, arterial (P < 0.05)

Po2; PacOz, arterial from

other

values

73+6 33+3 7.38rtO.03 12.7kO.3 152+16

Pco~; pH,, at that

work

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MUSCLE

2.

TABLE

0,

RESPIRATORY

REGULATION

AS

STUDIED

BY

transport and gas exchange during three conditions at rest and at two work intensities Stimulation Rest H

Muscle blood flow, ml 100 g-’ min-’ Cao,, ml/100 ml 0, delivery, ml 100 g-’ min-’ V02, ml. 100 g-l mid 0, extraction Pvoz, Torr Cvo2, ml/100 ml Pcoz, Torr DmOz 7 ml 100 g-l min-’ l

l

l

Intensity Maximal

3 Hz

I

C

38k7 6.2+0.4*

13+3* 14.220.5

29+6 14.6t0.5

2.4t0.5 0.4kO.2 0.18kO.05 22*1* 5.2+0.5*

1.8kO.4 0.6kO.l 0.36+0.04* 36-t3* 10.7kl.O

4.2t0.7* 0.6kO.l 0.15kO.02 45+3* 12.5kO.6

H

I

C

llOt8* 6.3*0.5*

35*7* 14.3k0.6

71&8* 14.6k0.6

137t8 6.3t0.5*

7.Okl.O 3.3kO.4 0.47?0.03* 18tl* 3.2&0.5*

5.1kl.l 2.61~0.5 0.55+0.07* 29+3 6.6*1.0*

10.3&0.7* 3.9kO.4 0.39+0.04* 33-t3 9.0&0.6*

8.7k1.2 4.520.5 0.53_+0.05* 16*2* 3.lt0.6* 19-t1* 0.26+0.04*

l

Values are means ? SE; IZ = 6. 0, delivery, arterial 0, content delivery; Pvoz , muscle effluent venous PO,; PEo2, calculated mean different (P < 0.05) from other values at that work intensity.

(CaO,) capillary

X muscle

blood flow; POT; DmOz, calculated

H

4.6t0.6

mM mM

13.OkO.6 2.9-0.2

V’J, mM

iPCrl/I pil mM

I

4.3kO.8 13.1kO.9 2.9kO.2

C

4.5t0.8 14.2+0.8*

3.1-t0.2 4.7kO.3 0.008+0.002

4.6k0.4

4.520.3

0.012+0.002

0.011+0.004

142228

149+34

146k25

7623

74+1

74*1

H

6.9t1.3 4.0k0.6 0.61kO.O6* 28+3 5.7*0.9* 40+3 0.11+0.02*

17.4&1.6* 7.4+0.7* 0.44+0.05* 32+4 8.6+0.9* 42k4 0.19+0.03*

116+10

mM-’

nM index,

Intensity

4.3kO.8

%

Values are means + SE; n = 6 (except for [ADP], [ATP]/[ADP][Pi], muscle tension. * Significantly different (P < 0.05) from other values

Maximal

I

C

4.5t0.6

H

5.8kO.8 2.OkO.4 0.017+0.003

5.7kO.8 2.OkO.2 0.022+0.005

4.7kO.5 2.7t0.2 0.015+0.002

44-tlO 95*10 91*3

3024

58kO.6 80+3 loot0

19+3 127+10 75*3

10.4kO.8

91*8

82-t5*

and [H+],, for which at that work intensity.

lization, and gas exchange. Muscle blood flow was significantly less during I at each stimulation level, as planned, and although muscle blood flow during H at the maximal stimulation intensity was 15% higher than during C (at the same perfusion pressure), this difference was not significant. At all three measurement periods, Qo, (muscle blood flow X arterial 0, content) was significantly greater during C, but at no time was Qo, significantly different between H and!, as planned. It should be noted, however, that at 3 Hz &o, was 27% less during I than during H (not significant), which resulted in proportionate VO, reduction and tension development impaired to a greater degree (Table 3). VO, was significantly greater during C only at the maximal stimulation intensity. The relationship between Qo, and VO, is illustrated in-Fig. 1. The PO, of the muscle effluent venous blood (Pvo,) was lower with H at both stimulation levels but was not different between C and I at the two levels. The calculated values of Dmo2 at the maximal stimulation rate showed significant differences among the three conditions, with the lowest Dmo2 during I and the highest during H. PCo2 at the maximal stimulation period was significantly less during H but was not different between I and C. The values related to muscle metabolism and respiration are presented in Table 3. The values of muscle [H+] (n = 4) were not significantly different among the three conditions at any measurement period, but they in-

4.7kO.7

I

4.4kO.5 7.2kO.8 8.5k0.3 0.9kO.l 0.027kO.005

10.3~05

[ATPI /[ADPI [PiI 7 Fatigue

14.820.5

muscle O2 uptake; 0, extraction, vo,/O, muscle 0, diffusing capacity. * Significantly

3 Hz

Rest

WI,,

48t9* 14.3kO.5

voq,

Stimulation

[ADP],

C

3. Muscle metabolism and respiration during three conditions at rest and at two work intensities

TABLE

[ATP], [PCr],

I

H

l

l

l

1665

31P-NMR

12.0*0.9*

n = 4). [H+],,

muscle

[H+];

Fatigue

C

4.2k0.6 8.1k1.3 8.5kO.4

4.320.6 7.9kl.O 8.5-t0.4

l.OkO.2

l.O-tO.2

0.022+0.006

0.032_+0.012

23k6 133t16 80+3

19-t4 115+19 91*3*

index,

ratio

of final

to maximal

creased significantly with work. These values of muscle [H+] are similar to that measured by the homogenate technique in this in situ muscle model during similar conditions [95 nM at rest to 125 nM during hypoxemic work (14)]. These [H+] values correspond to pH values of 7.02, 7.04, and 7.09 for H, I, and C, respectively, at 3 Hz, and 6.89, 6.87, and 6.94 at the maximal work intensity. The relatively small increase in muscle [H+] during work is likely a result of the lack of fast-twitch glycolytic muscle fibers in the dog gastrocnemius (21).

T ---A-

11

11

:

5

10

15

O2 delivery

(ml-100

g-’

.min

-+

20

-‘)

FIG, 1. Relationship between 0, delivery and muscle 0, consumption (VO,; means t SE) at 2 stimulation rates for 3 treatments: I, ischemia; H, hypoxemia; C, control.

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1666

MUSCLE

RESPIRATORY

REGULATION

Muscle [ATP] was never significantly different among conditions at rest or at either stimulation rate, and it never fell below resting concentrations even at the maximal stimulation level. The relationship between VO, and [PCr] is illustrated in Fig. 2. At rest and at 3 Hz, [PCr] was slightly greater during C than during H or I, but at the maximal stimulation level there were no significant differences in [PC,] among the three ’ conditions. For each of the three conditions there was a linear relationship between VO, and [PC,] (r = -0.99) over the three measurement periods, with the slopes significantly different between C and H or I but not between H and I. The relationship between [ PCr] /[Pi] and VO, is illustrated in Fig. 3. Figures 2 and 3 demonstrate the linearity of these relationships, with the slopes of H and I quite different from that of C but not different from each other. Calculated values of [ADP] were not significantly different at rest among the three conditions, and the increases with the stimulation rates were also not different among the three conditions. Figure 4 illustrates the relationships between VO, and [ADP]. As with Vo,/[PCr], \joJ[ADP] for C differed from that for H and I, but because muscle [H+] could only be accurately calculated in four of the animals, there is more noise in the [ADP] calculation than in the measured [ PCr]. Finally, the relationship between VO, and the phosphorylation potential [ATP] [ADPI-1 [Pi]-l, again calculated from four animals, is illustrated in Fig. 5. l

@=I

AS STUDIED

BY

31P-NMR

DISCUSSION

The results of this research demonstrated that when 0, delivery to working muscle was reduced by either ischemia or hypoxemia, the relationship observed between the variables believed to regulate respiration (such as ADP, Pi, and PCr) and tissue respiration (Vo2) was altered such that a greater regulatory signal was needed to elicit a given VO, (Figs. 2-5). When 0, availability to the working tissue is lowered (and presumably mean intracellular PO, is reduced), a stronger regulatory signal is required to drive mitochondrial respiration. These data suggest that 1) the sensitivity of mitochondrial respiration to these proposed regulators of tissue respiration can be modified by an interaction with tissue oxygenation, thereby indicating a regulatory role for 0,, and 2) under theconditions of this study, the bioenergetics of reducing VO, by hypoxemia or ischemia were similar at the same 0, delivery. 0, and tissue respiration. The regulation of tissue respiration has generally been considered to be controlled by the levels of the substrates needed to rephosphorylate ADP through oxidative phosphorylation, principally [ADP], [Pi], the [ATPI-to-[ADP][PJ ratio, and the redox ratio of NADH/NAD NADH

+ H+ + 3ADP + 3Pi + l/20, ~

l

IV

0

3ATP

O=H

O=H .=I

A=C

A=C

3

6

9

12

15

18

0.000

Wrl b-w FIG. 2. Relationship between concentrations of phosphocreatine ([PCr]) and muscle VO, (means & SE) at rest and during 2 work intensities for 3 conditions.

0.005

0.010

0.015

+ NAD + H,O

0.020

0.025

0.030

(0

0.035

CADPI (mM> FIG. 4. Relationship between [ADP] and muscle VO, (means at rest and during 2 work intensities for 3 conditions.

& SE)

10

I-

0 =H

O=H .=I A=C

T

l =I A =C

50

75

[ATP]/[ADP][Pi]

FIG. (means

3. Relationship between * SE) at rest and during

values of [PCr]/[Pi] 2 work intensities

and muscle Vo2 for 3 conditions.

FIG. (means

5. Relationship between + SE) at rest and during

100 (mM-

[ATP]/[ADP][Pi] 2 work intensities

125 ’)

and muscle VO, for 3 conditions.

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MUSCLE

RESPIRATORY

REGULATION

It has also been suggested (6) that another possible regulator of tissue respiration is the degree to which PCr is broken down, which might also serve as a signal linking the utilization of ATP in the cytosol with the production of ATP in the mitochondria. It has only recently been demonstrated (20, 26, 28, 31, 32) that the levels of some of these proposed regulators may be influenced by the amount of 0, available for tissue respiration, even when that available 0, is above that considered critical for tissue respiration. The results of the present study support those (12,20, 28) showing a role for 0, in modulating those factors believed to regulate tissue respiration. During conditions of either hypoxic or ischemic hypoxia in the present study, a greater concentration of the respiratory regulators (Figs. 2-5) was required to achieve a given VO,, as was seen in a prior study (12) during hypoxemia. These results demonstrate that the sensitivity of tissue respiration can be affected by tissue oxygenation. It was proposed in the prior study (12) that, as 0, delivery to the working tissue was reduced through hypoxemia, the mean intracellular PO, was reduced. This would be the result of a dynamic equilibrium established between the need for a high concentration of 0, at the mitochondria, so that the process described in Eq. 1 can proceed at a high rate, and the need to keep mitochondrial PO, low so that the diffusional gradient for 0, from capillary to mitochondria is maximized. In the present study, as 0, delivery was reduced, the mitochondrial PO, that was set by these two competing processes was likely also lowered. This may have resulted in some interaction between the reduced mean intracellular PO, and the other substrates of oxidative phosphorylation (Eq. I), so that the relationship between the amount of regulator needed to achieve a given tissue respiration was altered as a compensatory attempt to maintain Vo2. Although a small reduction in mean tissue PO, (even only l-2 Torr) may seem inconsequential, it should be remembered that, because of the shape of the oxymyoglobin dissociation curve, small changes in mean tissue PO, (at low mean tissue PO,) will cause large changes in myocyte [O,], because most of the myocyte [0,] is myoglobin bound. This change in mitochondrial sensitivity, possibly resulting from alterations in mean intracellular PO,, might occur through a mechanism similar to that shown when mitochondrial density is altered (10, 16): the amount of regulator needed to drive the mitochondria at a given respiratory rate is inversely related to the mitochondrial density. If the mean intracellular PO, was indeed reduced during the hypoxemic and ischemic conditions of the present study, it may have been analogous to the situation of reduced mitochondrial density (possibly through a lower amount of functional mitochondria), resulting in the change in mitochondrial sensitivity. Also, as Nioka et al. (22) suggested by use of a formulation of the Michaelis-Menten equation, if the potential maximum rate of ATP production via oxidative phosphorylation (V,,,) is not altered by reduced 0, availability, then, as PO, in the cell is diminished, the other regulators of oxidative phosphorylation must increase to maintain a given \j,z. However, Vmax may be interpreted as

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31P-NMR

the maximal amount of ATP turnover that can be attained for a given 0, delivery if all the 0, delivered to the working tissue is available to the mitochondria. As 0, delivery is decreased, the maximal amount of ATP turnover possible is diminished, and thus Vmax falls in proportion with 0, delivery (or that amount of 0, that can reach the mitochondria). As 0, delivery is diminished by either hypoxemia or ischemia, 0, extraction increases to some point determined by diffusional constraints (13, 14, 27) and tissue %,-to-blood flow heterogeneity (24). With Vmax falling with reduced 0, delivery, it can be seen from a modification of the Michaelis-Menten equation (2) vwIla2L = SI(K, + S) where v is tissue Tj,z, S is concentration of substrate, and K, is the Michaelis constant, as Vmax is reduced, S must increase to maintain v. If S is one of the regulators of oxidative phosphorylation (e. g., [ADP]), then, as Vmaxis reduced by decreased 0, availability, the [ADP] must increase to maintain a given VOW. This can be illustrated by use of a Hanes plot (Fig. 6), which uses the reciprocal form of the Michaelis-Menten equation, in which the Xintercept is the negative value of the K, for Pi/PCr (0.6), and the reciprocal slope of the line relating substrate (xaxis) and substrate-respiration (y-axis) is the estimate of Vmax.Figure 6 illustrates that the Vmag (reciprocal slope) of the mitochondria is indeed reduced more by hypoxemia and ischemia than by control conditions, with little difference between hypoxemia and ischemia (only one line drawn). This type of analysis suggests an 0, control of the mitochondrial Vmax so that, as the PO, at the mitochondria is reduced, the maximum rate of ATP formation via oxidative phosphorylation is also reduced. Finally, it should be noted that the proposed regulators of tissue respiration did not correlate strongly with 0, delivery; for example, at very different 0, deliveries and Tjoz values, almost identical values of [PCr] were found (Tables 2 and 3), indicating that it was likely that some intracellular factor, possibly changes in mean intracellular PO,, was modulating these regulators. In addition, extracellular hormonal levels were probably not factors influencing the altered mitochondrial sensitivity, inasmuch as these levels were likely quite different in hypoxemia and ischemia. -

0.30

z \ .-c 0.25-E b 0.20 -s V(v 0.15--

-0.6

O=H 0 =I n=c

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

[p~l/[pCrl FIG. 6. Hanes plot, using reciprocal form of Michaelis-Menten equation, showing relationship between substrate ([Pi] /[PC,]) and substrate-respiration ([Pi] /[PCr] lV0,). x-Intercept is -4, of substrate, and recipricol slope is estimate of mitochondrial Vmax.

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Hypoxemia and ischemia. An important observation from this study was that there was apparently no difference between hypoxemia and ischemia in the cellular bioenergetics (Figs. 2-5; although Fig. 5 seems to indicate a difference between hypoxemia and ischemia at 3 Hz, [ATP]/[ADP] [Pi] was a noisy calculation). 0, delivery to the working muscle was intentionally kept the same in hypoxemia and ischemia, with hypoxemia a high-flowlow-PaoZ situation and ischemia a low-flow-high-Paoz situation. Although there has been some speculation (29) that the reduction in iTo, during ischemia might be related more to the reduction in blood flow than to actual 0, availability and may be qualitatively different from reductions in VO, induced through hypoxemia, the results from the present study do not support this. Muscle blood flow during ischemia at both 3 Hz and the maximal stimulation intensity was reduced to 35% of the hypoxemic muscle blood flow, with no subsequent difference in muscle bioenergetics or Vo2. At the same 0, delivery, 0, extraction (VO,/QOJ was slightly higher (P < 0.05, Table 2) during the low-flow-high-Pao2 conditions of ischemia than during hypoxemia, as has been shown previously (14). More importantly, the relationship of VO, to the proposed regulators of respiration was the same for ischemia and hypoxemia, indicating that 0, lack in the tissue was likely similar under the conditions of this study. It might have been expected that mean tissue PO, would be lower in hypoxemia than in ischemia, because the PcoZ was so much lower in hypoxemia than in ischemia because of the difference between the two conditions in the Paog. Thus a lower tissue PO, would be necessary during hypoxemia to maintain a high O,-driving gradient from capillary to mitochondria. However, it appears that the higher flow during hypoxemia resulted in more capillary area available for 0, diffusion than in ischemia, thereby increasing the Dmoz of the tissue during hypoxemia (Table 2). It is likely that the mean tissue PO, was similar for hypoxemia and ischemia, because the reduced capillary surface area available for 0, diffusion during ischemia, and the resultant lower Dmo , meant that mean tissue PO, levels had &to fall very low during ischemia, as in hypoxemia, to offset the lower DmOz during ischemia and support maximal 0, flux into the cell. Finally, it should be noted that the contractions elicited in the current study were not extended for a time period long enough to investigate muscle fatigue performance. It is quite possible, as has been suggested (3), that the reduced-flow conditions of ischemia result in waste product accumulation that is different from that of the highflow conditions of hypoxemia, resulting in differences in long-term performance. In a previous study (12), ATP levels fell slightly during severe work in hypoxemic conditions, unlike the current study. Because of the manner in which the muscle had to be positioned within the magnet and the difficulty in securing the muscle in an optimally motionless position during the contractions, the tension developed in the present study was relatively low for the twitch contractions, resulting in a lower 0, extraction (compared with normal extractions by this muscle during reduced 0, delivery conditions) and the need for higher-than-normal twitch rates before fatigue set in. This might have af-

AS

STUDIED

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31P-NMR

fected the heterogeneity of blood flow to irO, and thereby altered the normal relationship between high-energy [Pi] and VO,. Although this makes it difficult to compare these results with those from previous studies in a quantitative fashion, the fact that the three conditions (control, hypoxemia, and ischemia) in this study were conducted in a repeated-measures design makes these results comparable within this study. Also, in the previous study (12) during hypoxemia (Pa,, = 20 Torr) comparable to the hypoxemia of the current work (Paoz = 24 Torr), the PCr-to-voZ ratio (1.83) at one work intensity was quite similar to that of the current study (1.60) at the maximal work rate. In addition, the differences in the calculated values of Dm,, for hypoxemic, ischemic, and control conditions in the present study are similar to those found in muscles exhibiting more normal extraction ratios during similar conditions (Hogan, unpublished observations). Although we postulate that the change in mitochondrial sensitivity that was found with the reduced 0, delivery of hypoxemia and ischemia was likely a result of some interaction with intracellular PO,, it should be noted that rates of fatigue were greater during hypoxemia and ischemia than during control conditions. If some fibers had quit working during hypoxemia and ischemia, rather than the whole muscle reducing tension development, then the VO, would be proportional to a smaller amount of tissue working and the amount of PCr measured to VO, produced might be more in line with that seen during control conditions. However, the final tension development of the muscle at the end of the maximal stimulation period was not significantly different among the three conditions, which would argue against this possibility. Also, at 3 Hz there was not much difference in fatigue development among the three conditions, with no significant difference in Vo2, yet PCr levels were significantly greater during control conditions than during hypoxemia and ischemia, and [ATP] /[ADP] [Pi] was close (P = 0.08) to being significantly greater during control conditions. At this time, it is difficult to separate the issue of 0, interaction with the regulators of tissue respiration from the possible effects of reduced fiber activation. Although we attribute the shift in mitochondrial sensitivity to alterations in tissue PO,, Gutierrez et al. (11) showed that PCr did not begin to decline during progressively increasing hypoxemia in resting rabbit muscle until critical levels of 0, delivery were reached and Tjoz began to fall. Nioka et al. (22) also showed little change in respiratory regulators (except for increasing NADH/ NAD) as brain tissue was made increasingly hypoxic until the VO, was compromised. Our data also show that, at rest, even with reduced 0, delivery in hypoxemia and ischemia and likely reduced intracellular PO,, the relationships of VO, to respiratory regulator were not different among hypoxemic, ischemic, and control conditions. It is possible that shifts in mitochondrial sensitivity caused by changes in tissue PO, occur only at higher rates of oxidative phosphorylation and when the tissue PO, falls below some critical level. Katz and Sahlin (20) showed that, at equal submaximal VO,, tissue metabolism and [PCr] were altered by hypoxemia, and Hogan and Welch (15) showed differences in muscle lactate

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MUSCLE

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REGULATION

concentration at equal 0, delivery and VO, when the Pa,, was varied. Limits to maximal respiration. It is likely that, for different systems (e. g., whole body human exercise compared with in situ muscle systems), any of the processes involved with 0, transport (lungs, heart, and blood characteristics), blood flow through the working tissue, gas and substrate diffusion into the celland to the mitochondria, diffusion of reactants (Eq. 1) throughout the cell, waste removal, and all the processes involved in excitation-contraction coupling, may under different conditions (e. g., before training compared with after) be an important limiting factor in determining the maximal VO, that can be attained. When 0, delivery is reduced to maximally working muscle, maximal 0, extraction from the reduced amount delivered is determined by the diffusing capacity of the tissue, the PO, gradient from capillary to mitochondria, and any heterogeneity of 0, delivery to Vo2 in the system. It is interesting to note that, at the highest VO, that could be achieved in the working muscle during the hypoxemic, ischemic, and control conditions of this study, there was no difference in the concentrations of the bioenergetic parameters (PCr, Pi, ADP, ATP, and ATP/ADP* Pi) measured, even though the VO, at the control condition was quite different from that at hypoxemia and ischemia. Although this would seem to support an 0, limitation under control conditions during the current study, inasmuch as the maximal bioenergetic level that could be achieved was the same as during the O,-limited conditions of hypoxemia and ischemia, it is difficult to assessthis possibility because of the poor tension development of the muscle and the subsequently poor 0, extraction and VO,. In a prior study (E), with more normal developed tension of the muscle, the PCr at the maximal stimulation intensity was significantly greater during control than during hypoxemic conditions. Therefore it is not yet possible to use these bioenergetic data as evidence of an 0, limitation at maximal stimulation rates during normal 0, delivery conditions. In the current study, ATP levels did not fall during the fatiguing contractions elicited, even though irO, was compromised, as was shown previously during slightly fatiguing work (12). It appears that working muscle has the ability to reduce the ATP utilization at the contractile sites, by reducing tension development, to that available from the ATP-regenerating sites, so that the total [ATP] of the cell does not fall (1). Through this control process, ATP breakdown and production are matched at rates that can be supported by the substrates needed for oxidative phosphorylation so that the cell [ATP] is well protected. Under these circumstances, it may appear that 0, levels in the tissue are above that considered critical for tissue respiration; however, this may be because the amount of 0, available for oxidative phosphorylation has through some regulatory process (possibly Pi inhibition of the contractile state) resulted in reducing ATP utilization to a rate that does not result in ATP depletion. Conclusions. The results of this research, measured using 31P-NMR, suggest a role for 0, availability at the mitochondria in adjusting their sensitivity to the proposed regulators of tissue respiration. This mav be be-

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BY 31P-NMR

cause fewer mitochondria are available for rephosphorylation as mean tissue PO, is decreased. These results support the notion that there may be a role for 0, in the regulation of tissue respiration, even at mitochondrial concentrations not considered rate limiting. Finally, these data also indicated no difference in muscle bioenergetics during the conditions of this study when irO, was reduced by either ischemia or hypoxemia at the same 0, delivery. The authors thank D. J. Wang for technical assistance in “‘P-NMR. This research was supported by National Heart, Lung, and Blood Institute Grants HL-17731, HL-180786, and HL-44125. M. C. Hogan is a Parker B. Francis Fellow in Pulmonary Research. Address for reprint requests: M. C. Hogan, Dept. of Medicine, 0623A, University of California, San Diego, La Jolla, CA 92093-0623. Received

25 October

1991; accepted

in final

form

15 May

1992.

REFERENCES 1. ARTHUR, P. G., M. C. HOGAN, D. E. BEBOUT, P. D. WAGNER, AND P. W. HOCHACHKA. Modeling the effects of hypoxia on ATP turnover in exercising muscle. J. Appl. Physiol. 73: 737-742, 1992. 2. BARBEE, R. W., W. N. STAINSBY, AND S. J. CHIRTEL. Dynamics of 02, CO,, lactate, and acid exchange during contractions and recovery. J. Appl. Physiol. 4: 1687-1692, 1983. 3. BARCLAY, J. K. A delivery-independent blood flow effect on skeletal muscle fatigue. J. Appl. Physiol. 61: 1084-1090, 1986. 4. CHANCE, B., S. NIOKA, AND J. S. LEIGH. Metabolic control principles: importance of the steady state reaffirmed and quantified by 31P MRS. In: Oxygen Transport and Utilization, edited by C. W. Byran-Brown and S. M. Ayres. Fullerton, CA: Sot. Crit. Care Med., 1987, p. 215-224. 5. CHANCE, B., AND G. R. WILLIAMS. Respiratory enzymes in oxidative phosphorylation. I. Kinetics of oxygen utilization. J. Biol. Chem. 217: 383-393, 1955. 6. CONNETT, R. J. Analysis of metabolic control: new insights using scaled creatine kinase model. Am. J. Physiol. 254 (Regulatory Zntegratiue Comp. Physiol. 23): R949-R959, 1988. 7. CONNETT, R. J., T. E. J. GAYESKI, AND C. R. HONIG. Energy sources in fully aerobic rest-work transitions: a new role for glycolysis. Am J. Physiol. 248 (Heart Circ. Physiol. 17): H922-H929, 1985. 8. CONNETT, R. J., AND C. R. HONIG. Regulation of Vo, in red muscle: do current biochemical hypotheses fit in vivo data? Am. J. Physiol. 256 (Regulatory Integrative Comp. Physiol. 25): R898-R906, 1989. 9. CONNETT, R. J., C. R. HONIG, T. E. J. GAYESKI, AND G. A. BROOKS. Defining hypoxia: a systems view of VO,, glycolysis, energetics, and intracellular PO,. J. Appl. Physiol. 68: 833-842, 1990. 10. DUDLEY, G. A., P. C. TULLSON, AND R. I. TEF~JUNG. Influence of mitochondrial content on the sensitivity of respiratory control. J. BioZ. Chem. 262: 9109-9114, 1987. 11. GUTTIEREZ, G., R. J. POHL, AND P. NARAYANA. Skeletal muscle 0, consumption and energy metabolism during hypoxemia. J. Appl. Physiol. 66: 2117-2123, 1989. 12. HOGAN, M. C., P. G. ARTHUR, D. E. BEBOUT, P. W. HOCHACHKA, AND P. D. WAGNER. Role of 0, in regulating tissue respiration in dog muscle working in situ. J. Appl. Physiol. 73: 728-736, 1992. 13. HOGAN, M. C., D. E. BEBOUT, P. D. WAGNER, AND J. B. WEST. Maximal 0, uptake of in situ dog muscle during acute hypoxemia with constant perfusion. J. Appl. Physiol. 69: 570-576, 1990. 14. HOGAN, M. C, J. ROCA, J. B. WEST, AND P. D. WAGNER. Dissociation of maximal 0, uptake from 0, delivery in canine gastrocnemius in situ. J. Appl. Physiol. 66: 1219-1226, 1989. 15. HOGAN, M. C., AND H. G. WELCH. Effect of altered arterial 0, tensions on muscle metabolism in dog skeletal muscle during fatiguing work. Am. J. Physiol. 251 (Cell Physiol. 20): C216C222, 1986. 16. HOLLOSZY, J. O., AND E. F. COYLE. Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. J. Appl. Physiol. 56: 831-838, 1984. 17. IDSTROM, J. P., V. H. SUBRAMANIAN, B. CHANCE, T. SCHERSTEN, AND A. C. BYLUND-FELLENIUS. Oxygen dependence of energy metabolism in contracting and recovering rat skeletal muscle. Am. J. Physiol. 248 (Heart Circ. Physiol. 17): H40-H48, 1985.

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18. JONES, D. P. Effect of mitochondrial clustering on 0, supply in hepatocytes. Am. J. Physiol. 247 (Cell Physiol. 16): C83-C89, 1984. 19. JONES, D. P., F. G. KENNEDY, AND T. Y. Aw. Intracellular O2 gradients and the distribution of mitochondria. In: Hypoxia: The Tolerable Limits, edited by J. R. Sutton, C. S. Houston, and G. Coates. Carmel, IN: Benchmark, 1988, p. 59-69. 20. KATZ, A., AND K. SAHLIN. Effect of decreased oxygen availability on NADH and lactate contents in human skeletal muscle during exercise. Acta Physiol. Stand. 131: 119-127, 1987. 21. MAXWELL, L. C., J. K. BARCLAY, D. E. MOHRMAN, AND J. A. FAULKNER. Physiological characteristics of skeletal muscles of dogs and cats. Am. J. Physiol. 233 (Cell Physiol. 2): C14-C18, 1977. 22. NIOKA, S. D., D. S. SMITH, B. CHANCE, H. V. SUBRAMANIAN, S. BUTLER, AND M. KATZENBERG. Oxidative phosphorylation system during steady-state hypoxia in the dog brain. J. Appl. Physiol. 68: 2527-2535, 1990. 23. PETROFF, 0. A. C., J. W. PRICHARD, K. L. BEHAR, D. L. ROTHMAN, J. R. ALGER, AND R. G. SCHULMAN. Cerebral metabolism in hyperand hypocapnia: 31P and ‘H nuclear magnetic resonance studies. Neurology 35: 1681-1688, 1985. 24. PIIPER, J. Unequal distribution of blood flow in exercising muscle of the dog. Respir. Physiol. 80: 129-136, 1990. 25. PIIPER, J., P. E. DIPRAMPERO, AND P. CERRETELLI. Oxygen debt and high-energy phosphates in gastrocnemius muscle of the dog. Am. J. Physiol. 215: 523-531, 1968.

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26. ROBIOLIO, M., W. L. RUMSEY, AND D. F. WILSON. Oxygen diffusion and mitochondrial respiration in neuroblastoma cells. Am. J. Physiol. 256 (Cell Physiol. 25): C1207-C1213, 1989. 27. ROCA, J., M. C. HOGAN, D. STORY, D. E. BEBOUT, P. HAAB, R. GONZALEZ, 0. UENO, AND P. D. WAGNER. Tissue 0, diffusion limitation of maximal 0, uptake in man. J. Appl. Physiol. 67: 291-299, 1989. 28. RUMSEY, W. L., C. SCHLOSSER, E. M. NUUTINEN, M. ROBIOLIO, AND D. F. WILSON. Cellular energetics and the oxygen dependence of respiration in cardiac myocytes isolated from adult rat. J. Biol. Chem. 265: 15392-15399, 1990. 29. STAINSBY, W. N., W. F. BRECHLJE, D. M. O’DROBINAK, A~~JD J. K. BARCLAY. Effects of ischemic and hypoxic hypoxia on VO, and lactic acid output during tetanic contractions. J. Appl. Physiol. 68: 574-579, 1990. 30. STAINSBY, W. N., AND H. G. WELCH. Lactate metabolism of contracting dog skeletal muscle in situ. Am. J. Physiol. 211: 177-183, 1966. 31. WILSON, D. F., M. ERECINSKA, C. DROWN, AND I. A. SILVER. The oxygen dependence of cellular energy metabolism. Arch. Biochem. Biophys. 195: 485-493, 1979. 32. WILSON, D. F., W. L. RUMSEY, T. J. GREEN, AND J. M. VANDERKOOI. The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration. J. Biol. Chem. 263: 2712-2718, 1988.

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