Role of 0, in regulating tissue respiration in dog muscle working in situ MICHAEL C. HOGAN, PETER G. ARTHUR, DONALD PETER W. HOCHACHKA, AND PETER D. WAGNER
E. BEBOUT,
Division of Physiology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623; and Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 2A9, Canada
as measured by 0, HOGAN,MICHAEL C.,PETER G. ARTHUR, DONALDE. BE- ase) activity] and tissue respiration, BOUT,PETERW. HOCHACHKA,AND~ETERD. WAGNER.ROZ~O~ uptake (VOW), this coupling of turnover to production is O2 in regulating
tissue respiration
in dog muscle working
in situ.
J. Appl. Physiol. 73(2): 728-736, 1992.-This study was designedto investigate the role of tissue oxygenation in someof the factors that are thought to regulate musclerespiration and metabolism. Tissue oxygenation was altered by reductions in 0, delivery (muscleblood flow X arterial 0, content), induced by decreasesin arterial PO, (Pa,J. 0, uptake (~oJ was measured in isolated in situ canine gastrocnemiusat rest and while working at two stimulation intensities (isometric tetanic contractions at 0.5 and 1 contractions/s) on three separate occasions,with only the level of Pa,, (78, 30, and 21 Torr) being different for each occasion. Muscle blood flow was held constant (pump perfusion) at each work intensity for the three different levels of Pa,,. Muscle biopsieswere obtained at the end of eachrest and work period. Muscle To2 was significantly less (P < 0.05) at both stimulation intensities for the hypoxemit conditions, whereas [ATP] was reduced only during the highest work intensity during both hypoxemic conditions (31% reduction at 21 Torr Pao2and 17% at 30 Torr). For each level of Pao2, the relationships between the changesthat occurred in VO, and levels of phosphocreatine,ADP, and ATP/ADP Pi as the stimulation intensity was increased were significantly correlated; however, the slopesand intercepts of these lines were significantly different for each Pao,. Thus a greater change in any of the proposed regulators of tissue respiration (e,g., phosphocreatine, ADP) was required to achieve a given VO, as Paqzwas decreased.These results indicate that VO, can be dissociatedfrom theseproposedregulators of tissue respiration and that this is likely due to an interaction with tissue or mitochondrial 0, tension, thereby demonstrating the importance of 0, as a modulator of the regulators of tissue respiration. l
considered to be very precise. Moreover, recent work has demonstrated that the cell ATP content remains nearly constant even at the highest work intensities, so there is never more than a small decrease in [ATP] (9,10,23,30). It seems that when ATPase rates are above the level that can be supported through rephosphorylation of ADP, the cell is capable of reducing ATPase activity (reduced tension development) to levels that do not allow substantial ATP depletion (1). In this way, it appears that the metabolic and respiratory regulatory processes of the cell are principally coupled to the maintainance of cellular ATP levels. This is likely important so that the membrane transport pumps, which require large amounts of ATP, sustain the ionic balance of the cell. The integration and regulation of the rephosphorylating processes to maintain ATP turnover are complex. For immediate regeneration of ATP, phosphocreatine (PCr) can be broken down by the creatine kinase reaction as follows MgADP-
+ PCr2- + H+ g MgATP2-
+ Cr
(0 where Cr is creatine, while oxidative phosphorylation in the mitochondria and glycolysis in the cytoplasm restore the majority of the ATP that is broken down during work. These processes are activated to different degrees by the intensity of the work, the time after the initiation of work, the duration of the work, the substrate available for the metabolic pathways, and 0, availability. The degree of activation of the rephosphorylating processes is thought to be controlled by the interaction of several biofatigue; skeletal muscle;gasexchange; exercise; acid-basebal- chemical parameters: concentrations of ATP, ADP, and ance; lactate; lactic acid; mitochondrial respiration; phospho- Pi; the phosphorylation potential as described by [ATP] / creatine [ADP] [Pi]; and the mitochondrial and cytoplasmic redox potential as described by [NADH] [H+]/[NAD]. In addition, it has also been suggested (4, 23, 27) that the most ALTHOUGH THE FACTORS that regulate and limit tissue important regulator of mitochondrial respiration may be respiration have been extensively studied, this area of the [PC,], inasmuch as it has been demonstrated that the amount of PCr broken down is proportional to VO, durresearch remains controversial and incompletely understood. At any given work intensity, there is a certain ATP ing the transition to submaximal work, at steady%tate turnover necessary to conduct the work required. This submaximal work, and during recovery. However, rather rate of ATP hydrolysis drives the complex processes that than there being a single regulator of the rephosphoryrephosphorylate ADP so that work can be continued at lating processes, it seems that different conditions can result in different interactions of these regulators. In an that particular rate. Because there is a strong correlation between work intensity [adenosinetriphosphatase (ATPextensive study of the regulation of respiration in in vivo 728
0161-7567192 $2.00Copyright 0 1992 the AmericanPhysiological Society
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.155) on December 14, 2018.
MUSCLE
RESPIRATORY
heart, From et al. (11) demonstrated that different levels of these regulatory parameters could be found at similar VO,, depending on the exogenous carbon source. Although the role of 0, in the process of maintaining [ATP] in the cell has generally been considered important only when [0,] is clearly rate limiting, Wilson and colleagues (32, 38) provided evidence that, even at submaximal and non-O,-limited rates of cellular respiration, [0,] may be an important modulator of these factors involved in respiratory regulation. It was the purpose of the present study to examine the relationships between the proposed regulators of tissue respiration and tissue VO, in working muscle when 0, delivery was altered so that the role of 0, in the factors involved in regulating tissue metabolism and respiration could be elucidated. METHODS
Six adult mongrel dogs of either sex (12-20 kg) were anesthetized with pentobarbital sodium (30 mg/kg), and maintenance doses were given as required. The dogs were intubated with cuffed endotracheal tubes, and ventilation was maintained with a Harvard 613 ventilator at a rate that achieved the desired PO, and Pco,. Esophageal temperature was maintained near 37OC by the use of heating pads. The animals were given heparin (1,500 U/ kg) after the surgery. Surgical preparation. The left gastrocnemius-flexor digitorum superficialis muscle complex (for convenience referred to as gastrocnemius) was isolated as described previously (17). Briefly, a medial incision was made through the skin of the left hindlimb from 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 artery (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 the ties. To prevent cooling and drying, all exposed tissues were covered with saline-soaked gauze and with a sheet of plastic wrap. After the muscle was surgically isolated, the Achilles tendon was attached to an isometric myograph (Statham 1360 transducer) to measure force development. The hindlimb was fixed at the knee and ankle and attached to the myograph with struts to minimize movement. Weights were used at the end of each experiment to calibrate the force myograph.
REGULATION
BY
0,
729
The isometric force developed by each muscle was normalized to the weight of that muscle and is reported as a “developed tension” (N/100 g tissue). Isometric muscle contractions (tetanic) were elicited by stimulation of the sciatic nerve with square-wave impulses (4-6 V) of 0.2-ms duration for 200 ms at 50 Hz. The muscle was stimulated at 0.5 contractions/s for 3 min and was then stimulated at 1 contraction/s for 3 min. A stimulation rate of 1 contraction/s achieves the maximal VO, for this in situ muscle model (33), 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 a tension setting of -10 g force per gram muscle mass (estimated before the experiment) was recorded. This ensured that the initial tension development was not affected by slippage in the system that might have occurred during the prior contraction period. This resting muscle length was slightly less than the length at which the contractile response was greatest. Experimental protocol. Before the first contraction period, the blood supply to the isolated muscle was switched from self-perfusion to pump perfused and enough time was allowed for conditions to stabilize at a blood flow similar to the self-perfused level. All blood flows were measured from the venous outflow by use of a graduated cylinder to collect the blood for 30-s. Each experiment (n = 6) consisted of three separate contraction periods for the isolated muscle. Each contraction period, consisting of the 3-min submaximal and 3-min maximal stimulation rates, was separated by 15-20 min of rest and was different only in the fraction of inspired 0, that the dog breathed to produce the three different values of arterial PO, (Pa,,). During the rest period preceding and during each contraction period, the dog breathed 21, 10, or 7% 0, in N, to produce the desired Pa,,. The order of the treatments was varied so that all six possible orders of the three treatments were conducted once. Muscle blood flow was set at a level that achieved a perfusion pressure of -120 Torr during the first contraction period, at both the submaximal and maximal stimulation level (which caused a higher flow in the maximal stimulation condition). The same blood flow was used in the subsequent contraction periods, so that muscle blood flow was matched in all three Pa,, conditions for both the submaximal and maximal stimulation rates. Measurements. Arterial blood samples from the arterial line entering the muscle 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. Muscle biopsies were obtained during the same time periods that the blood measurements were taken. These biopsies were obtained using a rapid-freezing biopsy drill (model 950B, Alko), which was able to freeze the sample in liquid N, in
E. BEBOUT,
Division of Physiology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623; and Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 2A9, Canada
as measured by 0, HOGAN,MICHAEL C.,PETER G. ARTHUR, DONALDE. BE- ase) activity] and tissue respiration, BOUT,PETERW. HOCHACHKA,AND~ETERD. WAGNER.ROZ~O~ uptake (VOW), this coupling of turnover to production is O2 in regulating
tissue respiration
in dog muscle working
in situ.
J. Appl. Physiol. 73(2): 728-736, 1992.-This study was designedto investigate the role of tissue oxygenation in someof the factors that are thought to regulate musclerespiration and metabolism. Tissue oxygenation was altered by reductions in 0, delivery (muscleblood flow X arterial 0, content), induced by decreasesin arterial PO, (Pa,J. 0, uptake (~oJ was measured in isolated in situ canine gastrocnemiusat rest and while working at two stimulation intensities (isometric tetanic contractions at 0.5 and 1 contractions/s) on three separate occasions,with only the level of Pa,, (78, 30, and 21 Torr) being different for each occasion. Muscle blood flow was held constant (pump perfusion) at each work intensity for the three different levels of Pa,,. Muscle biopsieswere obtained at the end of eachrest and work period. Muscle To2 was significantly less (P < 0.05) at both stimulation intensities for the hypoxemit conditions, whereas [ATP] was reduced only during the highest work intensity during both hypoxemic conditions (31% reduction at 21 Torr Pao2and 17% at 30 Torr). For each level of Pao2, the relationships between the changesthat occurred in VO, and levels of phosphocreatine,ADP, and ATP/ADP Pi as the stimulation intensity was increased were significantly correlated; however, the slopesand intercepts of these lines were significantly different for each Pao,. Thus a greater change in any of the proposed regulators of tissue respiration (e,g., phosphocreatine, ADP) was required to achieve a given VO, as Paqzwas decreased.These results indicate that VO, can be dissociatedfrom theseproposedregulators of tissue respiration and that this is likely due to an interaction with tissue or mitochondrial 0, tension, thereby demonstrating the importance of 0, as a modulator of the regulators of tissue respiration. l
considered to be very precise. Moreover, recent work has demonstrated that the cell ATP content remains nearly constant even at the highest work intensities, so there is never more than a small decrease in [ATP] (9,10,23,30). It seems that when ATPase rates are above the level that can be supported through rephosphorylation of ADP, the cell is capable of reducing ATPase activity (reduced tension development) to levels that do not allow substantial ATP depletion (1). In this way, it appears that the metabolic and respiratory regulatory processes of the cell are principally coupled to the maintainance of cellular ATP levels. This is likely important so that the membrane transport pumps, which require large amounts of ATP, sustain the ionic balance of the cell. The integration and regulation of the rephosphorylating processes to maintain ATP turnover are complex. For immediate regeneration of ATP, phosphocreatine (PCr) can be broken down by the creatine kinase reaction as follows MgADP-
+ PCr2- + H+ g MgATP2-
+ Cr
(0 where Cr is creatine, while oxidative phosphorylation in the mitochondria and glycolysis in the cytoplasm restore the majority of the ATP that is broken down during work. These processes are activated to different degrees by the intensity of the work, the time after the initiation of work, the duration of the work, the substrate available for the metabolic pathways, and 0, availability. The degree of activation of the rephosphorylating processes is thought to be controlled by the interaction of several biofatigue; skeletal muscle;gasexchange; exercise; acid-basebal- chemical parameters: concentrations of ATP, ADP, and ance; lactate; lactic acid; mitochondrial respiration; phospho- Pi; the phosphorylation potential as described by [ATP] / creatine [ADP] [Pi]; and the mitochondrial and cytoplasmic redox potential as described by [NADH] [H+]/[NAD]. In addition, it has also been suggested (4, 23, 27) that the most ALTHOUGH THE FACTORS that regulate and limit tissue important regulator of mitochondrial respiration may be respiration have been extensively studied, this area of the [PC,], inasmuch as it has been demonstrated that the amount of PCr broken down is proportional to VO, durresearch remains controversial and incompletely understood. At any given work intensity, there is a certain ATP ing the transition to submaximal work, at steady%tate turnover necessary to conduct the work required. This submaximal work, and during recovery. However, rather rate of ATP hydrolysis drives the complex processes that than there being a single regulator of the rephosphoryrephosphorylate ADP so that work can be continued at lating processes, it seems that different conditions can result in different interactions of these regulators. In an that particular rate. Because there is a strong correlation between work intensity [adenosinetriphosphatase (ATPextensive study of the regulation of respiration in in vivo 728
0161-7567192 $2.00Copyright 0 1992 the AmericanPhysiological Society
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.155) on December 14, 2018.
MUSCLE
RESPIRATORY
heart, From et al. (11) demonstrated that different levels of these regulatory parameters could be found at similar VO,, depending on the exogenous carbon source. Although the role of 0, in the process of maintaining [ATP] in the cell has generally been considered important only when [0,] is clearly rate limiting, Wilson and colleagues (32, 38) provided evidence that, even at submaximal and non-O,-limited rates of cellular respiration, [0,] may be an important modulator of these factors involved in respiratory regulation. It was the purpose of the present study to examine the relationships between the proposed regulators of tissue respiration and tissue VO, in working muscle when 0, delivery was altered so that the role of 0, in the factors involved in regulating tissue metabolism and respiration could be elucidated. METHODS
Six adult mongrel dogs of either sex (12-20 kg) were anesthetized with pentobarbital sodium (30 mg/kg), and maintenance doses were given as required. The dogs were intubated with cuffed endotracheal tubes, and ventilation was maintained with a Harvard 613 ventilator at a rate that achieved the desired PO, and Pco,. Esophageal temperature was maintained near 37OC by the use of heating pads. The animals were given heparin (1,500 U/ kg) after the surgery. Surgical preparation. The left gastrocnemius-flexor digitorum superficialis muscle complex (for convenience referred to as gastrocnemius) was isolated as described previously (17). Briefly, a medial incision was made through the skin of the left hindlimb from 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 artery (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 the ties. To prevent cooling and drying, all exposed tissues were covered with saline-soaked gauze and with a sheet of plastic wrap. After the muscle was surgically isolated, the Achilles tendon was attached to an isometric myograph (Statham 1360 transducer) to measure force development. The hindlimb was fixed at the knee and ankle and attached to the myograph with struts to minimize movement. Weights were used at the end of each experiment to calibrate the force myograph.
REGULATION
BY
0,
729
The isometric force developed by each muscle was normalized to the weight of that muscle and is reported as a “developed tension” (N/100 g tissue). Isometric muscle contractions (tetanic) were elicited by stimulation of the sciatic nerve with square-wave impulses (4-6 V) of 0.2-ms duration for 200 ms at 50 Hz. The muscle was stimulated at 0.5 contractions/s for 3 min and was then stimulated at 1 contraction/s for 3 min. A stimulation rate of 1 contraction/s achieves the maximal VO, for this in situ muscle model (33), 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 a tension setting of -10 g force per gram muscle mass (estimated before the experiment) was recorded. This ensured that the initial tension development was not affected by slippage in the system that might have occurred during the prior contraction period. This resting muscle length was slightly less than the length at which the contractile response was greatest. Experimental protocol. Before the first contraction period, the blood supply to the isolated muscle was switched from self-perfusion to pump perfused and enough time was allowed for conditions to stabilize at a blood flow similar to the self-perfused level. All blood flows were measured from the venous outflow by use of a graduated cylinder to collect the blood for 30-s. Each experiment (n = 6) consisted of three separate contraction periods for the isolated muscle. Each contraction period, consisting of the 3-min submaximal and 3-min maximal stimulation rates, was separated by 15-20 min of rest and was different only in the fraction of inspired 0, that the dog breathed to produce the three different values of arterial PO, (Pa,,). During the rest period preceding and during each contraction period, the dog breathed 21, 10, or 7% 0, in N, to produce the desired Pa,,. The order of the treatments was varied so that all six possible orders of the three treatments were conducted once. Muscle blood flow was set at a level that achieved a perfusion pressure of -120 Torr during the first contraction period, at both the submaximal and maximal stimulation level (which caused a higher flow in the maximal stimulation condition). The same blood flow was used in the subsequent contraction periods, so that muscle blood flow was matched in all three Pa,, conditions for both the submaximal and maximal stimulation rates. Measurements. Arterial blood samples from the arterial line entering the muscle 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. Muscle biopsies were obtained during the same time periods that the blood measurements were taken. These biopsies were obtained using a rapid-freezing biopsy drill (model 950B, Alko), which was able to freeze the sample in liquid N, in
