:1, M ‘ 3512:... dz .. {I 3 f)... K; h. .L . . .: llillllllm \\\\\\\\\\\\\\\\\\\\\\\\\\\\ This is to certify that the dissertation entitled Control of Aerobic Metabolism in Skeletal Muscle presented by Susan Jill Harkema has been accepted towards fulfillment of the requirements for Ph.D. Physiology degree in \ y . , Major professor Date 7/17/‘7? l Mun-m. Arr r . A --_I .An . , . . 0.1m: _ LIBRARY M'chan State University *— PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE CONTROL OF AEROBIC METABOLISM IN SKELETAL MUSCLE BY Susan Jill Harkema A DISSERTATION Submitted to Michigan State University 'in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1993 ABSTRACT CONTROL OF AEROBIC METABOLISM IN SKELETAL MUSCLE BY Susan Jill Harkema The primary focus of this research was to test the two prominent models of the control of respiration in skeletal muscle. The two models are: 1) simple kinetic control, or control by total cytoplasmic ADP and 2) thermodynamic control, or control by cytoplasmic phosphorylation potential. These tmwi hypotheses are cdifficult to distinguish by simple correlative experiments because both ADP and cytoplasmic phosphorylation potential change in tandem chnflmm; muscle stimulation. However, it luxi been shown that the study of contracting muscles during experimentally induced acidosis could” in principle distinguish between the two models. Therefore, the primary research objective was to examine the effects of acidosis on respiratory control during submaximal ATPase rates in skeletal muscle. Although there is general agreement that respiration is controlled by "phosphorylation state” (either ADP, or cytoplasmic phosphorylation potential) in most skeletal muscles, there was some evidence that phosphorylation state is not time sole controller (Hf respiration ljl slow-twitch muscle at higher respiration rates. Therefore, the initial focus (Hf this research was t1) re—investigate this phenomenon in intact slow—twitch muscle. 0W3 examined the relationship between phosphorylation state and oxygen consumption in cat soleus muscle in situ. One series of experiments Hwnitored EKkn ATP, EU. and {MT noninvasively during submaximal stimulation in intact soleus muscles using 31P—NMR. Another series measured oxygen consumption during steady—state conditions at identical stimulation rates. The results did not confirm the previous studies and suggested that phosphorylation state :hs the dominant regulator of respiration in slow-twitch muscle. The remainder of the research focused on distinguishing between the twwu models of tflua control of respiration by experimentally manipulating the intracellular concentration of hydrogen ions .A potential obstacle to the design of the this study was the possibility that acidosis alters the utilization of ATP, as well as ATP synthesis. For example, if acidosis profoundly inhibits cross-bridge cycling, ii; would kxe difficult to increase respiratory rate by muscle stimulation in acidic muscles. Therefore, the purpose of the second study was to directly measure the effect of hypercapnic acidosis on ATP utilization during isometric contractions of perfused cat fast— and slow—twitch muscles. ATP utilization was observed during acidosis using gated 31P-NMR if} isolated cats soleus and biceps muscles. The results showed that the ATP cost of tetanic contractions is reduced in proportion to the reduction in force. Thus, the intrinsic rate of cross-bridge cycling and the economy of force development appeared not to be sensitive to lowered pH. The final series of experiments were then designed and implemented ix) test time control <3f respiration 1]) slow— twitch muscle. We examined the effect of hypercapnic acidosis on the relationship between PCr, ADP, and phosphorylation potential versus respiratory rate in intact cat soleus muscle at rest and during moderate stimulation. Intracellular pH was decreased by changing the gas content of the perfusate, metabolite concentrations (PCr, Pi, ATP, and pH) were measured by 31P—NMR, and oxidative rates were calculated from oxygen consumption measurements in the slow—twitch muscles. Although interpretation of the study was complicated by the observation that acidosis decreased the maximum aerobic capacity of muscle, the results were clearly not consistent with the simple ADP model of respiratory control, but did remain consistent with thermodynamic :models. We conclude that the control of respiration is regulated by cytosolic phosphorylation potential and not by ADP availability in skeletal muscle. Copyright by SUSAN JILL HARKEMA 1993 I dedicate this thesis to my family and friends who made this possible: Mom and Dad, Julie, Tommy, and Sallie, your continued love, support, and encouragement never ceases and for that I am truly fortunate; Kate and Todd, you've seen me through the tough times, and of course, the good times; Glenna and Marsha, you've always accepted who I am and believed in me; Steve and Linda, you were there when I most needed you, and of course, your funny; Van, your patience, trust, understanding, and love is a miracle. vi ACKNOWLEDGMENTS I would like to thank Dr. Ronald Meyer for providing me with the opportunity to do exciting research and learn innovative technology under his direction. I am extremely fortunate to have you as a mentor and most admire you for your straightforward, intelligent approach to science and your pure quest for fully supported scientific answers. iI greatly appreciate time contributions of Imy thesis committee members. Dr. Adams your encouragement and guidance was very valuable to me. There are so many students over the years who have benefited from your wisdom and I feel very fortunate to be one of them. Dr. Chaudry, I especially thank you for giving me the encouragement to pursue graduate work. Dr. Jump, I have learned many aspects of becoming a successful scientist by your example and only hope I can reach the consistent level of hard work and perseverance you maintain every day. Dr. Hollingsworth, I appreciate your participation on my committee and your insightful suggestions. Dr. Krier, your motivational talks and sound scientific advice were very valuable in my early development as a scientist. I would like to sincerely thank the members of the RAM lab for there support. Dr. Greg Adams, thanks so much for vH your "million" ideas, inn: most important if; that almost every one was a good one. Your contribution to my work was significant and I look forward to future collaboration during (nu: careers. Dr. Jeanne Foley, II appreciate your contributions to my graduate experience and especially enjoyed time "weekend“ experiments. Mary' Peterson, your technical assistance was extremely valuable as well as your support. Tony Paganini, you always kept things exciting, good luck in the completion of your thesis. Rob MacDonald, your work in the lab and on my projects was outstanding. I am sure your career as a physical therapist and researcher will be a successful one. I would like to thank all my fellow graduate students for their support and commitment to success. I would especially .like t1) acknowledge [MEL Samita IBhattacharya, Jon Feeman, Ormond MacDougald, and Zhawoen Wang for their encouragement, sharing of ideas, and "intellectual discussions", all which greatly enhanced my graduate school experience. Finally, I would like to thank Sharon Shaft for all she did for me from the very first day of graduate school. Also my heartfelt thanks to Barb Burnett, Bobbi Milar, Marilyn Walker and Shirley Zutaut for their invaluable assistance and incredible support over the years. vm TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES I. II. INTRODUCTION AND LITERATURE REVIEW SKELETAL MUSCLE METABOLISM OXIDATIVE PHOSPHORYLATION THEORIES OF CONTROL OF OXIDATIVE PHOSPHORYLATION Inorganic Phosphate (Pi) ADP Cytoplasmic Phosphorylation Potential [ATP]/[ADP] Creatine APPLICATION OF PHOSPHORUS NUCLEAR MAGNETIC RESONANCE STUDIES TO MUSCLE METABOLISM 31P-NMR Studies Supporting Regulation By Redox Potential 31P-NMR Studies Supporting ADP Control xii xiii 11 15 17 20 26 27 29 33 34 31P—NMR Studies Supporting Thermodynamic Models 37 THE PHOSPHORYLATION STATE CONTROVERSY SUMMARY RESEARCH OBJECTIVES GENERAL MATERIALS AND METHODS PHOSPHORUS NUCLEAR MAGNETIC SPECTROSCOPY(§lP-NMR) 45 46 48 49 49 General Principles NMR Probes For Muscle Studies Procedures For The Basic 31P—NMR Pulse Experiment Gated 31P—NMR Pulse Experiment 49 56 69 7O III. IV. VI. MUSCLE PREPARATIONS In situ Soleus Studies Isolated Perfused Muscle Studies CONTROL OF RESPIRATION BY PHOSPHORYLATION STATE IN SLOW-TWITCH MUSCLE IN SITU INTRODUCTION MATERIALS AND METHODS Surgical Techniques Experimental Series I/Phosphorus NMR Experimental Series II/Oxygen Consumption RESULTS DISCUSSION EFFECT OF HYPERCAPNIC ACIDOSIS ON THE ATP COST OF CONTRACTIONS IN FAST- AND SLOW-TWITCH MUSCLES INTRODUCTION MATERIALS AND METHODS Surgical Techniques Gated 31P-NMR Experiments RESULTS DISCUSSION CONTROL OF RESPIRATION IN SLOW-TWITCH MUSCLE INTRODUCTION MATERIALS AND METHODS Surgical Techniques Experimental Series I/Phosphorus NMR Experimental Series II/Oxygen Consumption RESULTS DISCUSSION SUMMARY AND CONCLUSIONS 71 74 76 83 83 86 86 87 88 89 105 109 109 112 112 113 114 125 128 128 133 133 133 135 135 149 153 Table Table Table Table Table Table LIST OF TABLES Mammalian Skeletal Muscles. PCr time constants. pH, force, oxygen consumption, and blood flow measurements of soleus muscle at rest and during stimulation. Biceps and soleus muscle phosphate levels and contractile properties during hypercapnia and normocapnia. PCr time constants. Phosphate levels, pH and oxygen consumption in soleus muscle during hypercapnia and normocapnia. m 94 98 116 139 140 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 10. ll. 12. 13. LIST OF FIGURES Schematic of muscle contraction. Schematic of Oxidative Phosphorylation. Linear Circuit Model Representation of the Free Induction Decay (FID) and NMR spectrum Chemical structure of PCr, ATP, and Pi and the phosphorus NMR spectrum containing the corresponding peak that represents each phosphate signal. Plexiglas/Lexan probe constructed for animal experiments within a 4.7 Tesla magnet. Schematic of Helmholtz coil and circuitry of the coil. Circuit for specially constructed force transducers. Probe for isolated perfused muscle experiments within a 9.2 Tesla magnet. Top portion of probe for isolated perfused muscle experiments within a 9.2 Tesla magnet. Gated 31P—NMR Experimental Protocol. Isolated perfused muscle experimental setup. Series of spectra of soleus muscle, control (1 minute), (15 minutes), and during recovery xH during 3 Hz stimulation (15 minutes). 13 4O 52 54 59 61 63 66 68 73 82 91 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 14. 15. 16. 17. l8. 19. 20. 21. 22. 23. 24. PCr levels from 31P-NMR spectra of cat soleus muscle acquired during and after 15 minutes of stimulation at 0.5, l, 2, 3, and 4 Hz. PCr time constants for stimulation rates of 0.5, l, 2, 3, and 4 Hz. Steady—state oxygen consumption vs. of stimulation rate times mean peak twitch force during soleus muscle stimulation. Steady-state PCr vs. rate times mean peak twitch force during soleus muscle stimulation. Relationship between steady-state PCr and oxygen consumption at rest and during stimulation (0.5, 1, 2, 3, 4 Hz). Spectra acquired at rest and immediately following isometric twitch and tetanic contractions in soleus muscle during normocapnia and hypercapnia. Spectra acquired at rest and immediately following isometric twitch and tetanic contractions in biceps muscle during normocapnia and hypercapnia. Energy cost (umol PCr/g muscle) per isometric contraction during normocapnic and hypercapnic perfusion of soleus and biceps muscles. Energy cost and force production per isometric contraction during normocapnic and hypercapnic perfusion of soleus and biceps muscles. PCr changes during soleus muscle stimulation and recovery under normocapnic and hypercapnic conditions. pH changes during 0.25 and 0.5 Hz stimulation of soleus muscle during hypercapnia and normocapnia. xm 93 97 product 100 product of stimulation 102 104 118 120 122 124 138 142 Figure 25. Figure 26. Figure 27. [ADP] versus oxygen consumption at 144 0.25 Hz and 0.5 Hz during hypercapnia and 0.25 Hz, 0.5 Hz, and 1 Hz during normocapnia. Steady-state PCr levels versus oxygen 146 consumption of soleus muscle during rest and stimulation at 0.25 and 0.5 Hz during hypercapnia and 0.25, 0.5, and 1 Hz during normocapnia Cytoplasmic phosphorylation potential 148 versus oxygen consumption of soleus muscle during rest and stimulation at 0.25 and 0.5 Hz during hypercapnia and 0.25 Hz, 0.5 Hz, and 1 Hz during normocapnia. xiv "M“ ‘ ‘ “disguisigju. ..2 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW The pathways of intermediary metabolism and ATP production in muscle and other tissues are known in remarkable detail. However, our understanding of the control mechanisms of these pathways is often surprisingly primitive. This discrepancy between our knowledge of the biochemical machinery, versus our understanding of how this machinery actually works If} intact tissues, ix; perfectly illustrated when considering the control of respiration in skeletal muscle. As outlined below, the potential regulatory mechanisms linking respiratory rate 11) muscle work were identified by the early 1960's. By 1980, the general mechanisms by which mitochondria synthesize .ATP were clear, although some molecular details are still not complete. Nonetheless, the exact signal which links mitochondrial adenosine triphosphate (ATP) synthesis to cytoplasmic adenosine triphosphatase (ATPase) rate in muscle is still a major source of controversy among muscle physiologists. The experiments described 1J1 this thesis will help to cflarify this issue, at least (1) the extent that. they' seem t1) eliminate CNN? major‘ hypothesis, i.e., that muscle respiration. is regulated in 51 simple Michaelis/Menton fashion by total concentration. The following discussion will: 1) overview of muscle metabolism; 2) the various proposals for time contnil of respiration skeletal muscle, including the adenosine diphosphate (ADP) control hypothesis, and 3) rationale for the experiments included in this thesis. SKELETAL MUSCLE METABOLISM The primary energy utilizing functions of skeletal muscle cells are to maintain cell homeostasis and to perform mechanical work. As in other tissues, cell homeostasis is maintained by several energy utilizing pumps that regulate transmembrane ionic concentrations and intracellular calcium levels (Janis et al., 1987), and by protein synthesis and other basic anabolic reactions. In addition to these basic functions, skeletal muscle performs work following stimulation of the myocyte by a motor neuron. Electrical current travels along t-tubules changing the transmembrane potential. The sarcoplasmic reticulum releases bound stores of calcium which diffuse to contractile proteins, actin and myosin (Hasselbach and Oetliker, 1983; Donaldson and Kerrick, 1975). These proteins overlap and calcium binds to troponin exposing the binding site on actin for myosin. Crossbridges form and cytoplasmic ADP provide a general consider in some detail in introduce the methods and use energy for movement, leading to crossbridge cycling and ultimately muscle contraction [Figure 1] (Cooke, 1990; Huxley and Simmons, 1971; Phillips and Petrofsky, 1983). Whether the energy is used for cell homeostasis or crossbridge cycling, .ATP ii; the energy' source (Lipmann, 1946, Cain and Davies, 1962). ATPases within the myoctye cleave the terminal phosphate of ATP to release it's stored energy. Furthermore, the products of ATP hydrolysis, ADP and inorganic phosphate (Pi), are well known activators of the major ATP synthetic pathways in the cell i.e., glycolysis, and oxidative phosphorylation (Alberty, 1968; Lehninger, 1954). Thus, ii: seems .reasonable 13) suppose that these products, or some function of them, may provide the feedback signal controlling ATP synthesis. This basic assumption underlies all major theories of respiratory control, at least in skeletal muscle, and. is no doubt generally correct. As we will see below, the main controversy is over exactly which product, or combination of products, is operationally the effective controller in intact muscle. There is a net decrease in chemical potential energy during muscle activity with ATP hydrolysis (Lipmann, 1949; Rall, 1972), yet ATP itself rarely decreases significantly during muscle contraction because it is rapidly rephosphorylated by the creatine kinase reaction [Equation 2]. This reaction has been proven to be near thermodynamic Figure 1. Schematic of muscle contraction. AL) s11) _ ® '5‘ ’1“’. ""\\’ will 5 ‘\ ’,$\ 14_’ {91". — Tropomyoein Thick fiament (myosin) (ED (ED (ED -‘ '7‘- i’K‘ ”\‘ ”\\ I, Iflliil ”'5‘ M!!!“ J . ~ In‘a:ai.u9; .c-. N.” -;- ...l _ 1 Thin iilancnt Aotin IIIIIIllIlllllllllllIIIllIlllllllllIIll-Illaln===:*-——————" “muss; .- equilibrium in intact muscle (McGilvery and Murray, 1974; Lawson and Veech, 1979) and provides access to a phosphate pool for rapid ATP regeneration. Hence, even in the absence of other ATP synthetic reactions, the observed chemical reaction which drives muscle contracthmi is not net ATP hydrolysis per se, but rather net hydrolysis of PCr to creatine and Pi: ATP + H20 :3.ADP + Pi + dH+ (ATPase) [1] ADP + PCr + H+ C3 Cr + ATP (Creatine Kinase) [2] PCr + BH+ :3 Cr + Pi [3] B = (l - a) is 0.4 at pH 7.0 and 0.65 at pH 6.5 (Gilbert et al., 1971; Meyer and Adams, 1990, Adams et al., 1990). The effect is that changes in ATP are effectively buffered by PCr during contraction by nearly instantaneous rephosphorylation cflf ADP (Bienfait. et al., 1975; Mahler, 1985). However, because the level of ADP in muscle at rest is so low (<50 umol), even an insignificant change in ATP can still result in significant accumulation of ADP. Furthermore, the net reaction does release Pi, so there is a significant change in the free energy of ATP hydrolysis as PCr is depleted: AG = AGO' + 2.303 RT log [ADP][Pi][Hi] [4] [ATP] AGO' = -30.5 kJ/mol (Chang, 1981); R=8.314 J/K/mol. Thus, muscle contraction and ATP use is accompanied by significant increases III ADP, and IPi enmi a significant decrease in cytosolic free energy of ATP hydrolysis, even though ATP does not significantly change. Free cytoplasmic ADP is run: easy to measure in Hmscle, because the basal level is low, and because there is a large pool of ADP bound to actin and other proteins which is released during extraction for biochemical assays. Some evidence suggests that chemical measurements of EM. in muscle are inaccurate for similar reasons. However, PCr is easy to measure. As we will see below, non-invasive measurements of PCr by NMR spectroscopy are now commonly used as ea general index of muscle energy cm: ”phosphorylation state", and ere specifically, can be lfififii to calculate free ZHH> and the free energy of ATP hydrolysis in muscle. Finally, before proceeding to the details of various models for respiratory control, it should be noted that mammalian skeletal muscle is not a biochemically homogenous tissue. There are at least three muscle fiber types characterized by different glycolytic, oxidative, and myosin ATPase activities [Table 1] (Close, 1972). Resting Pi levels are higher and PCr and ATP levels are lower in slow-twitch as compared to fast-twitch fibers at rest (Meyer et al., 1985; Meyer et al., 1982; Crow and Kushmerick, 1982). Also, ATPase rates and oxygen consumption rates are lower in slower contracting muscles fibers compared to tflufi: of faster contracting fibers at 1 Actomyosin ATPase .Activity Glycolytic Capacity Oxidative Table 1 MAMMALIAN SKELETAL MUSCLES FAST—TWITCH EAST-TWITCH SLOW-TWITCH GLYCOLYTIC OXIDATIVE— OXIDATIVE GLYCOLYTIC High High Low High High High Low High High identical twitch rates (Meyer et al., 1985). Velocity of shortening, as time classification. implies, is nuufli more rapid 1J1 fast—twitch fibers (Claflin anui Faulkner, 1985) but slow-twitch fibers are more fatigue resistant (Baldwin and Tipton, 1972). Myosin proteins are distributed differently between the fiber types with high concentrations CH? Type I III SICWJ muscle fibers and, in contrast, primarily Type IIa and IIb in fast muscle fibers (Close, 1972). These differences must be kept in mind for two reasons: first, it is conceivable that respiration is controlled differently in different fiber types; and second, these differences complicate the interpretation of studies performed on muscles of mixed fiber types. OXIDATIVE PHOSPHORYLATION Oxidative metabolism is the major source of ATP produced during muscle stimulation at rates below the maximum aerobic capacity of muscle (Erecinska and Wilson, 1982; Kushmerick, 1983; Mahler, 1985). The following discussion briefly outlines the history and major conclusions of studies of oxidative phosphorylation. Engelhardt was time first ix) demonstrate ea fluoride- insensitive, cyanide-sensitive respiratory-linked synthesis of ATP from inorganic phosphate distinct from glycolysis in bird erythrocytes in the early 1930's (Engelhardt, 1932). Yet, oxidative phosphorylation did not seem to become 10 accepted until several years later when Kalckar showed phosphorylation CH? glucose, glycerol and 19H? coupled. to respiration (Kalckar, I937; Kalckar, 1939). Several investigators showed that respiratory—linked phosphorylation is fundamentally different from glycolysis and. requires ;phosphate acceptor (ADP), and Pi (Johnson, 1941; Ochoa, l940a; Ochoa, 1940b; Belitzer and Tzibakowa, 1939). The discovery (M? the reactions of time Kreb's cycle explained the stimulation of respiration which is observed with addition of dicarboxylic acids and demonstrated that NADH, the product of the dehydration of these substrates, is directly oxidized by the respiratory chain. This clearly established involvement of the respiratory chain in oxidative phosphorylation of ADP (Slater, 1981). Lehninger verified. this link tn/ demonstrating" phosphorylation accompanies passage of electrons from NADH to oxygen in rat liver mitochondria (Lehninger, 1954; Lehninger and Wagner Smith, 1949). Chance and Williams (1955; 1956) introduced an assay method. to :measure oxidative phosphorylation in isolated mitochondria and then outlined the sequence of the respiratory chain components. They measured rapid changes in respiration caused by addition of ADP in isolated mitochondria using a special double beam spectrophotometer. They , 4) [ATP]/[ADP] ratio, and 5) creatine via the ”creatine shuttle". 1. Inorganic Phosphate (Pi) Johnson (1941) first noted the importance of both Pi and phosphate acceptor as essential reactants of both aerobic and anaerobic breakdown of carbohydrate. In the late forties and early fifties, Lardy and colleagues (1951), demonstrated that availability of either Pi or 16 phosphate acceptors profoundly effects oxidatiwe rate of several substrates in isolated rat liver mitochondria and also showed that the effects were reversible. Rat liver mitochondria displayed an impressive increase in respiration with availability of phosphate acceptor. Creatine and partially purified creatine kinase provided a phosphate acceptor system that increased respiration 10- fold. Ihi all cases excess phosphate acceptor and enzyme were added. With B-hydroxybutyrate as substrate there was no apparent oxygen consumption until phosphate acceptor systems or dinitrophenol were added (Lardy, 1951; Lardy and Wellmen, 1951). In summary, Johnson proposed that oxidative rates are limited by rate of hydrolysis of high energy phosphates and their synthesis is coupled to oxidative electron transport. The "P potential" was identified as important in directing "oxidative systems” (Lardy and Wellmen, 1951). They argued that inorganic phosphate is the key factor in respiratory control, although with low sensitivity, since respiration continued at 51 reduced rate iflmai phosphate vmms depleted. Because Pi may also be an important factor in control of glycolysis, this scheme was attractive. If Pi regulates both glycolysis and respiration, this would describe a very simple and efficient scheme for metabolic control (Lardy anmi Wellmen, 1951; Lardy and lMaley, 1954; Lardy, 1951; Lynen, 1942; Kingsley-Hickman et al., 1987; Johnson, 1941; Lardy and Wellman, 1953). 2. ADP Chance and VHlliams (1956) challenged time idea that phosphate controlled respiration and proposed that ADP was the "physiological substance" responsible for activation of oxidative phosphorylation with stimulation. They developed a rapid assay for oxidative phosphorylation that measured respiration caused by addition of known concentrations of ADP in isolated guinea pig and rat liver mitochondria (Chance and Williams, 1955). In these experiments respiration. rate increased. rapidly vniji the addition. of exogenous ADP followed by a decrease to a resting state as ADP is exhausted. Ttmn/ estimated that the luichaelis constant for ADP control of mutochondria was 20 - 30 um from measurements of deceleration of respiration as a bolus of added ADP was depleted. Also, in the course of these experiments, they defined the iinn: classic states (If respiration. States 22 and 5 were characterized by extremely low rates of oxygen consumption, with state 2 created by lack of organic substrate, and state 5 being created by oxygen deprivation. State 4 is aerobic with excess oxygen and organic substrate, but with no or very low ADP. This is the state which presumably corresponds to the resting state in skeletal muscle. State 3 is the active state with adequate supplies (of all substrates. In. state 13 a steady-state level of cytochrome c is maintained while ADP varies tremendously. 18 The respiratory control ratio was defined as the ratio of maximum oxygen consumption in state 3 divided by that in state 4L .A wide range of respiratory control ratios were observed, ranging from 6 in ascite tumor cell mitochondria to 65 in guinea pig liver mitochondria. It was concluded that LMMV ratios indicated damaged mfltochondria III which electron transport was uncoupled from ATP synthesis, whereas high ratios indicated functionally intact mitochondria (Chance, 1959). Chance (1959) pointed out that it vanUi be difficult to estimate the respiratory control ratio of mutochondria in intact liver cm: tumor cells, since tflrfis would require experimental control of the total cellular ATPase rate. In contrast, it is easy to vary the work and ATPase rates of skeletal. muscle- cells tux electrical stimulation. Thus, Chance recognized that skeletal muscle is an excellent tissue for studies of respiratory control, and ever since then muscle has been the main focus of his work. Chance and Connelly (1957) identified ADP as the activator of oxidative phosphorylation in muscle cells following a contraction. They estimated increments of ADP or Pi concentration by measuring oxidation of pyridine nucleotides. Repeated treasurements were inade CH1 intact muscle and the sensitivity to ADP was less than 0.001 mol/g with a response time of less than 0.1 seconds. They published numerous studies (if isolated mdtochondria from various sources, including skeletal muscle, afld- of which 19 showed similar sensitivity to added ADP, with Km in the 20 - 50 um range, which is approximately the ADP level estimated in intact skeletal muscle (Chance, 1959; Chance et al, 1962; Chance, 1965). Chance and coworkers recognized the possibility that Pi also plays a role in controlling respiration but discounted. it CH1 several grounds. Firsts they' reasoned that the level of Pi in resting muscle, as measured by chemical analysis was well above the Km of mitochondria for Pi (Chance, 1959). Thus, increases in Pi could not account for the large increases in oxygen consumption measured during Imuscle stimulation. In. contrast, they' estimated that only 6% of total ADP released with a twitch is required to stimulate respiration half-maximally in muscle (Chance and Williams, 1955). Second, they pointed out that in yeast and ascites tumor cells transition from state 4 to 3 is achieved by addition of glucose is associated with an increase ir1.ADP and rm) change ijt Pi (Chance anmi Maitra, 1963). Finally, in many publications, as mentioned previously, they point out that Km (20—50 MM) estimated in isolated mitochondria is reasonably close to the levels of ADP in skeletal muscle during moderate exercise. Since the pioneering work of Chance and coworkers, many other studies of both isolated mitochondria and intact tissues have been interpreted in terms of this simple model of kinetic control of respiration by ADP levels, and this is the only ‘view typically' :mentioned. in standard 2O biochemistry and physiology texts. Despite the development of the alternative models discussed below, there is little doubt that under some conditions, respiration in isolated mitochondria can be made to behave as predicted by the ADP model. A good illustration of this is the work of Jacobus (1985; 1982) idm) defended time classical ZEN) availability model and further contended that neither ATP/ADP ratio nor cytoplasmic phosphorylation potential (see below) correlated with respiration rate. hm these experiments, steady—state conditions were established in a solution containing isolated rat liver mitochondria and various amounts of ATP and Pi. Hexokinase, a ADP generating enzyme was added in various amounts to titrate respiratory rates. Only ADP concentrations consistently correlated with respiration rate under afld. the conditions studied, providing' good. evidence for JUN? regulatirmx of oxidative phosphorylation. 3. Cytoplasmic Phosphorylation Potential Early on, Klingenberg (1961) argued that respiratory control could not be explained by simply ADP or Pi limitation. He observed respiration tm> be dependent on [ATP], and reported an ATP—dependent increase in the apparent Km of oxidative phosphorylation for ADP. He introduced the thermodynamic equilibrium hypothesis, with cytoplasmic phosphorylation potential (ln([ATP]/[ADP][Pi])) (Veech et al., 1979) as the parameter that actually 21 determines rate of respiration. This theory purports that an approximate thermodynamic equilibrium exists between the pyridine nucleotide pool, the electron transport chain, and high energy phosphate pools. According to this view, respiratory chain enzymes respond to changes in cytoplasmic phosphorylation potential, generated kn/ various ATPases (H? the cell. A near equilibrium. between the respiratory chain and .ATP synthesis would provide emu efficient and precise control mechanismt for (generation (Hf ATP. The "best evidence in favor (If near‘ equilibrium] control (Hf respiration :hs the observation that the reactions of the respiratory chain are reversible, i.e., that under some conditions ATP hydrolysis by the mitochondrial ATPase can drive reverse flow in the respiratory? chain (Lardy enmi Wellmen, 1951, jKlingenberg, 1961). If equilibrium control models are correct, then the free energy Lost tn/ any rmflur of electrons from. reduced pyridine nucleotide (NADH) to cytochrome a3 should be quantitatively recovered by phosphorylation of 3 ADP molecules. The overall chemical equilibrium reaction is: NADH + 2a3+3 + 3ADP + 3Pi ¢:> NADI + 2a3+2 + 3ATP. [5] 22 and the equilibrium constant of this reaction: [NAD+] [a3+2]2 [ATP]3 [6] [NADH] [a3+3]2 [ADP]3[Pi]3 Electron flow generates ATP related to the difference between oxidation—reduction potentials of pyridine nucleotide, Eh(NAD), enmi cytochrome exp Eh(a3). Energy from electrons flowing from NADH to cytochrome a3 will be equal to three times the free energy of ATP synthesis by the conservation (n? free energy (Owen anmi Wilson, 1974). This does not require that phosphorylation potential in the mitochondria and cytoplasm must be identical but only that translocation occurs without significant loss of free energy (Wilson et al., 1974). Thus, thermodynamic theory declares that the free energy's of cytosolic ATP hydrolysis and the oxidation—reduction reaction should be equivalent. Gibb's free energy of ATP hydrolysis is given by: AG = AGO' + 2.303RT log [ADP][Pi][Hi] [7] [ATP] (AGO' = -30.5 kJ/mol (Chang, 1981); R=8.3l4 J/K/mol. Cytosolic phosphorylation potential is derived from this equation anmi is considered El marker CH? changes 1J1 free energy potential of the adenylate system. The free energy change of oxidation-reduction reactions is given by: AG = —nFAE [8] (n=# (If electrons transferred Emu: molecule, E‘== 96,486.7 C/mol; TAE == potential difference between the donor and acceptor redox couples (Chang, 1981). Hassinen. and Hiltunen (1975) supplied evidence for near—equilibrium between the phosphorylation state of adenine nucleotides and redox state of respiratory carriers using surface fluorometry and spectrophotometry in beating and arrested rat hearts. KCl induced cardiac arrest caused a reduction CM? fluorescent flavoproteins anmi nicotinamide nucleotides, oxidation of cytochromes a enmi c, inhibition of respiration, and increased cytoplasmic phosphorylation potential. The difference between the beating and arrested heart of AE of ATP hydrolysis was 21.2 mV comparing favorably with at 23 mV change in [H2 of NAD/NADH and the cytochrome chain. These results demonstrated that cytochromes c and a are in near equilibrium with the state of cytoplasmic adenylates, thus supporting the equilibrium hypothesis (If mitochondrial respiratory control iii intact myocardial tissue. The most widely cited studies in favor (NE a form of near—equilibrium control are those of Wilson and colleagues (Wilson 6%: al., 1974; Erecinska 6N: al., 1974; Holian 6% al., 1977; Owen and Wilson, 1974; Erecinska et al., 1977). In brief, their conclusions rest on the following series of observations. First, in isolated mitochondria, they found an excellent agreement between the calculated 24 intramitochondrial free energy potentials of NAD redox reactions and time extramitochondrial free energy (Hf ATP hydrolysis. Second, they found the same agreement in calculated potentials derived from metabolite measurements in intact liver cells. Third, in experiments similar in design to those reported by Jacobus and colleagues (Jacobus and Lehninger, 1973; Jacobus et al., 1982; Jacobus, 1985), they found that only phosphorylation potential consistently correlated with respiration rate. The reason for the discrepancy 1J1 results between the studies (Hf Wilson and colleagues and those of Jacobus are difficult to explain, since the experimental designs were quite similar. Van Der Meer (1978) applied the principles of non— equilibrium thermodynamics to the study of respiratory control. Although formally different that the above equilibrium theories, in general outline the conclusion is similar, that the rate of oxidative phosphorylation ought to depend on the free energy difference between mitochondrial metabolites and cytoplasmic phosphates (Van Der Meer 6%: al., 1980; Van [Mun et al., 1980). In these studies, cytosolic phosphorylation potential was measured and NADH/NAD was calculated from lactate/pyruvate and 3-8 hydroxybutyrate/acetoacetate ratios in perfused isolated liver" cells iii steady~stater conditions (Van Ikn: Meer' et al., 1978). The respiratory chain was considered a "black box" catalyzing the overall reaction IQ 'J‘ NADH + H+ + 3ADP + 3P1 + 1/202 CD NAD+ + 3ATP +4H20. [9] An affinity term was defined dependent on NADH/NAD, [ADP][Pi]/[ATP],and OZ. Steady oxygen consumption was found to be linearly dependent on this affinity term and they concluded oxygen consumption is regulated by cytosolic phosphorylation potential, mitochondrial redox potential and partial pressure of oxygen. A basic prediction of all these thermodynamic models of the control of respiration. is that the forward and reverse flux through the mitochondrial ATPase ought to be nearly equal, and both fluxes should be greater than any net flux. This prediction was examined in isolated mitochondria by LaNoue and coworkers (1985). Unidirectional rates were measured tux radioactive tracer methods over a range of respiratory rates. At rates near State 4, the fluxes were nearly equal and larger than the net flux. However, as respiration rate was increased toward maximum (State 3), the reverse flux (ATP —e ADP + Pi) decreased. At maximum state 3 rate, the forward flux (Pi + .ADP -e’ ATP) was nearly equal to the net flux. Similar results were obtained by Ugurbil and colleagues in working rat hearts using NMR spin-transfer methods. Based on these studies, it appears that near equilibrium theories of control may apply at relatively low respiratory rates, but rmm: at near meximum rates, when kinetic limitations become dominant. 4. [ATPJ/[ADP] Slater also supported the thermodynamic hypothesis (Bienfait. et al., 1975) Ibut later suggested that [ATP]/[ADP] ratio, independent (N5 Pi was time controlling parameter (Slater et al., 1973). This theory is based on the idea that time adenine translocator, which transports ATP and ADP across the mitochondrial membrane, (Klingenberg, 1980; Vignais, 1976), limits respiration (Vignais, 1976; Letkc> et al., 1980; Kunz. et al., 1981; Kuster 6%: al., 1976). In contrast II) the thermodynamic theories, this assumes that the translocator is displaced far from equilibrium, although within the mitochondria, the nucleotides may cm: may not tme near equilibrium vniii the respiratory chain (Kuster et al., 1981; Erecinska and Wilson, 1982; Kunz et al., 1981). Holian and Wilson (1977) argued against the translocase theory by showing that [Pi] altered respiration rate in isolated mitochondria when the [ATP]/[ADP] remained constant. Oxidative phosphorylation increased by 230 percent with constant [ATP]/[ADP] and a four—fold increase in [Pi], and increases up ti) 4000 percent were cmeerved with maximum Pi stimulation. Moreover, they studied mitochondria with different respiratory control ratios (RCR). As the RCR decreased the dependence on Pi decreased. Thus timn/ proposed timm: during' isolation. at the mutochondrial inner membrane, 28 and therefore the rate of respiration, is limited by the diffusion of creatine to the mitochondria. Mahler (1985) developed at mathematical. model of respiratory control based CH1 the creatine shuttle. This model satisfactorily explained time observathmd that both PCr and oxygen consumption change over a mono—exponential time course after a step change in work rate. Based on the assumption that respiration rate is linearly proportional ti) mean cytoplasmic creatine levels, ZMahler‘ derived. the following equation relating changes in oxygen consumption to changes in either creatine or PCr: AQ02(t) = —1/Ip A[PCr](t) = l/Ip A[creatine](t). [10] [PCr] and [creatine] are total content per unit weight, AQ02(t), A[PCr](t), A[creatine](t) are changes in their respective values from basal levels, I is the time constant of AQOZ’ and p is the P/02 ratio for oxidative metabolism, expected to be 6 mol/mol (Lemasters, 1984, Crow and Kushmerick, 1982, Chance and Williams, 1955). The creatine shuttle model has been criticized by several researchers. Meyer (1984) demonstrated that this model is really a special case of the more general phenomenon CM? facilitated diffusion. Model calculations showed that, although much of the intracellular transport of high energy phosphate in normal muscle must be carried by PCr and creatine, this is simply a consequence of the 29 fact that ATP hydrolysis and synthesis occur at different places in the cell. Once equilibrium of creatine kinase is acknowledged, this transport effect cannot kme offered as evidence timm: creatine irs mechanistically rmnme important than ADP itself. In addition, several studies have shown that ATP and ADP can diffuse freely between myofibrils and mitochondria without the assistance of the creatine kinase reaction (Yoshizaki et al., 1987; Yoshizaki et al., 1990). Further studies demonstrated that muscle depleted of creatine and PCr by chronic feeding of creatine analogs are capable of normal, or even better than normal, aerobic metabolism (Shoubridge and Radda, 1987; Meyer, 1989). Taken together these studies demonstrate that creatine is not required for the control of respiration in muscle. Finally, as shown below, the result of Mahler's mathematical analysis can tme reproduced beginning with a completely different mechanistic assumption, assuming that cytoplasmic ZHT> free energy controls respiration (Meyer, 1985). APPLICATION OF PHOSPHORUS NUCLEAR MAGNETIC RESONANCE STUDIES TO MUSCLE METABOLISM Hoult (1974) and coworkers published phosphorous Nuclear Magnetic Resonance (31P-NMR) spectra of intact biological tissues, opening the ckmnr for study (Hf muscle 3O metabolismttxy this uniquely non—invasive technique. They identified peaks corresponding ti) Pi, ATP, Emu? and sugar phosphates 1J1 intact rat immmflirm> muscle. This vmus an important discovery for subsequent investigations of skeletal muscle metabolism, because now free levels of high energy phosphates could kme measured repeatedly 1J1 intact tissue during rest, stimulation, and recovery without disruption of the tissue (Dawson et al., 1980; Taylor et al., 1986; Meyer et al., 1982; Ingwall, 1982) Among the first results of 31P-NMR studies in skeletal muscle was the recognition that resting metabolite levels in muscle were somewhat different than those measured chemically after tissue extraction (Meyer et al., 1985). Early chemical analysis had given variable results for skeletal muscle PCr, and especially Pi concentrations. The most careful investigators detected lower levels of Pi (Seraydarian tet al., 1961), but :H: wasn‘t timid. 31P-NMR spectroscopic results became available that the relatively lower level of EM. (2-3 mM versus 8-10 rmm was generally accepted. The difference between chemical and NMR measurements could be attributed to artifactual hydrolysis of PCr during the extraction procedure in some cases (Meyer et a1. 1985) However the possibility that ea significant fraction CM? total EU. hi muscle is kxnnmi to intracellular proteins, and therefore not visible in high resolution spectra, has not been ruled out. In either case, it is now clear that free, metabolically active Pi in intact muscle, especially fast~twitch muscle, is less than measured chemically (Meyer et al., 1985; Meyer et al., 1982; Kushmerick and Meyer, 1985). 31P-NMR can investigate the intracellular environment and allow calculation of energetic parameters such as [ADP] or cytoplasmic phosphorylation potential (Meyer et al., 1982). Relative areas are measured and compared to ATP metabolite levels, which remain. relatively! stable: during chemical analysis. Transient metabolic events can be followed using 31P—NMR at rest, and during stimulation and recovery in skeletal muscle. Intracellular creatine kinase enzymes were confirmed to kme in chemical equilibrium, in intact muscle, by using NMR spin transfer techniques (Gadian et al., 1981). A novel feature of 31P—NMR measurements is that they allow non—invasive estimates of intracellular hydrogen ion concentration. Intracellular pH was first measured by 31P— NMR iii erythrocytes (Moon anmi Richards, 1973) and subsequently NMR became an important non—invasive tool to monitor til changes ll] cell suspensions (Gillies est al., 1981) and intact tissues (Meyer et al., 1982; Balaban, 1984; Dawson et al., 1980). pH measurement depends on the fact that the resonant frequency (chemical shift) of Pi is sensitive to pH changes within the biological range of ph 6.2—7.4. On the other hand, PCr is insensitive to hydrogen ion changes in this range since it's pKa is 4.5. Thus PCr provides a stable chemical shift reference for muscle b.) Ix) studies. Titration curves enme established tin: phosphate chemical shifts in solutions identical in ionic strength, temperature and metabolite concentration, to the intracellular milieu of skeletal muscle. Although the accuracy of the method depends on independent estimates of such factors, intracellular pH (mni be measured 1J1 intact striated muscle with a very high degree of precision by NMR. Of course, the git measured depends CH1 the intracellular localization of the phosphate observed. There is good evidence that this is predominantly from the cytoplasmic compartment ii11muscle tissue (Bailey 6%: al., 1981). Intracellular free magnesium concentration can also be determined by observing the chemical shifts of ATP peaks (Hoult et al., 1974; Gupta and Moore, 1980). Over the last decade there have been. many 31P-NMR studies of skeletal and heart muscle (Meyer et al., 1982; Arnold et al., 1984; Ingwall, 1982; Balaban, 1984; Katz et al., 1987; Argov et al., 1986; Dawson et al., 1977; Jacobus et al., 1977; Sapega em: Assuming temperature of 37°C, 1 mM magnesium concentration, pH = 7.1, 5 mM ATP concentration, ch = 1.3 x 10'9 (Lawson and Veech, 1979), and Km = 2 x 10‘5 M (Chance and Williams, 1956): .A._ — [l4] Vmax 1 + (0.52/([Cr]/[PCrl)) 31P—NMR can detect levels of Pi but not Cr. However, levels of Cr and Pi are approximately equal in resting muscle and during mild exercise (Kushmerick and Meyer, 1985; Meyer et al., 1985; Meyer and Adams, 1990). By simple substitution: : [15] Vmax 1 + 0.52/([Pi]/[PCr]) Thus, the transfer function between work, V/Vmax, and the biochemical response, Pi/PCr, is expected to approximate a rectangular hyperbola with "Km" 0.5 to 1 (Chance et al., 1986; Leigh et al., 1986). Many published studies of both animal and lunmni muscle) appear ti) support. this analysis (Chance EN: al., 1986; Nioka 6%: al., 1992; Chance en: al., 1981; Chance et al., 1992; Yoshizaki et al., 1987). Chance and coworkers also used 31P—NMR to re- investigate respiration Ill isolated mdtochondria 1J1 order to bridge the gap between previous invasive studies and their more recent studies of intact cells (Gyulai et al., 1985). Substrates and oxygen were added to the suspensions to maintain steady~state respiration and examine phosphates between respiration states 4 and 3. They compared relationships of Pi/PCr ratio, cytoplasmic phosphorylation potential, and free energy of hydrolysis of ATP. linear relationships were observed with all parameters dependent on ATPase rate. Nonetheless, the authors attributed oxidative control to ADP availability alone, and concluded that time same interpretation could kme applied tx) all of their in vivo studies (Arnold et al., 1984; Partain et al., 1984; Chance et al., 1992; Chance et al., 1981; Leigh et al., 1986). 31P-NMR Studies Supporting Thermodynamic Models Meyer and coworkers published a series of 31P-NMR studies which, in aggregate, seem to support thermodynamic theories of respiratory control (Meyer, 1989; Foley et al., 1991). Interpretation of these studies relies on a circuit analog model (M? the relation between changes iii PCr and respiration in muscle which is described below (Meyer 1988). Hill (1940a) was the first to report a simple exponential time course for oxygen consumption during recovery after a single tetanus. Mahler, using a different method (if measurement also :fimumi oxygen consumption. to follow this same pattern (Mahler, 1985). Mahler was surprised to observe this simple response, since it suggested oxidative phosphorylation is limited by a single step that follows an apparent first—order rate law, despite the fact that the scheme of reactions involved are complex. Thus, Mahler derived the model mentioned above by assuming that respiration depends directly on cytoplasmic creatine. Meyer's model is based on an entirely different mechanism, namely thermodynamic control. However, time mathematical result is very similar to Mahler's. The linear model of the control of respiration (Meyer, 1988) vmms derived from time principles cof non—equilibrium thermodynamics with a 20—fold concentration range among tissues. Thus, the results are applicable for many muscle types, ranging from smooth to fast-twitch muscle. He concluded that the ADP and cytosolic phosphorylation potential models can not be distinguished by simple correlative experiments in which steady—state metabolite levels are compared to steady-state 46 oxygen consumption. These parameters are highly correlated because .ADP enmi phosphorylation. jpotential. are Iboth ultimately derived from measurement of PCr. Connett went on to show that discrimination between these models may occur tux changing cnme system variable QiL total adenine content, % phosphorylation of creatine) independent of changes in stimulation rate in intact tissue. In particular, he showed that the ADP model might be tested by independently altering intracellular pH. This suggestion formed the rational for the third experiment described in this thesis (Connett, 1988b; Funk et al., 1990; Connett and Honig, 1989; Connett et al., 1990). SUMMARY The putative controllers of oxidative phosphorylation within the aerobic capacity of striated muscle are redox potential and phosphorylation state. Evidence 1J1 cardiac tissue suggests that redox potential may play an important role. In skeletal muscles most evidence suggests phosphorylation state as the dominant controller. However, despite 40 years of research, the exact mechanism of phosphorylation state control in intact muscle is still not clear. Chance and Williams introduced the hypothesis based on enzyme kinetics that solely' [ADP] concentraticmi was the controlling factor. Models of control by ATP/ADP, or by 47 creatine, are mechanistically similar, and lead to similar experimental predictions. Klingenberg first introduced the idea of thermodynamic control, suggesting cytosolic phosphorylation potential ([ATP]/[ADP][Pi]) is the critical regulatory signal. Wilson supported this theory and then later expanded the model to include redox potential and oxygen availability. Meyer eventually reduced the thermodynamic model to a circuit analog, which predicts the metabolic behavior of muscle during stimulation at submaximal rates, when organic substrates and oxygen are not limiting. Thus, the two prominent proposed signaling mechanisms are [ADP] and cytosolic phosphorylation potential. It is possible to test these models by defining steady-state relationships between oxygen consumption and these metabolites while independently varying pH (Connett, 1988a; Connett and Honig, 1989). The primary objective of this dissertation. is to test these two competing" models for control of respiration by phosphorylation state. As noted above, there ire some evidence that phosphorylation state is not the sole controller of respiration in slow—twitch muscle at 'higher respiration rates. Therefore the initial focus of this research was to re-investigate this phenomenon in intact slow-twitch muscle. These experiments are GOOO FID l l /’n/e/25/'f/ >11 frequency Spectrum Figure 5. Chemical structure of PCr, ATP and Pi and the phosphorus NMR spectrum containing the corresponding peak that represents each phosphate signal. 54 D=Q—|O n:.< ZOE do :0 old]? :0 55 nucleus, and lme allowed ti) relax completely back ii) the original orientation to detect a maximum signal on the next pulse. This relaxation (characterized by a time constant, T1) typically takes 10—15 s for phosphorus nuclei in biological tissues. If a submaximal pulse is given (<90°) or only partial relaxation of the nuclei occur, only a fraction of the signal is observed and saturation of the signal. occurs. Molecular' interactions affect relaxation times and therefore nuclei of different molecules relax at different rates. This may cause variable signal saturation unless the delay between scans is long enough to allow all nuclei to fully relax. Unfortunately, multiple FID's or "scans" typically must be acquired and averaged together to achieve a reasonable signal to noise ratio because the sensitivity of NMR is relatively low. The signal to noise ratio increases with the square root of the number of acquired scans. For example, ti) acquire 16 scans vuth.1£) s between scans (to allow full relaxation) would take 4 minutes. However, if repeated scans are applied too closely together, only partial relaxation will occur between scans and the peaks become partially saturated. This is (Hme of the :major technical problems in quantitative in vivo NMR studies. Some of our experiments required less than one minute time resolution, therefore we used.ee method developed to reduce the saturation effects, yet also improve time 56 resolution. The NMR signal can be maximized even with a reduction in time between scans (termed relaxation delay) by giving 21 shorter pulse width (~60fl. Some saturation still occurs, therefore, vwa optimized (nu: parameters to obtain the best S/N ratio for primary signal of interest, the PCr peak. When quantitative comparisons between different peak areas were required, we corrected for saturation effects by comparing peak areas in fully—relaxed (15 :3 pulse interval) enmi saturated CL — 13 s interval) spectra of resting muscle. NMR Probes For Muscle Studies An NMR study of Hmscle requires a strong homogenous magnetic field, a radio—frequency transmitter and receiver, and apparatus to measure physiological parameters such as temperature and muscle force. The experiment must be done if} the :magnet, and therefore, commercial apparatus that contains magnetic materials cannot be used because they would destroy the homogeneity of the magnetic field. Furthermore, any wires to or from recorders or stimulators must be filtered to prevent other rf signals (TV or radio stations) from entering the magnet. Standard chemistry NMR "probes" supplied with NMR equipment do not contain enough space for this extra apparatus. Finally, because the experiments require time best possible S/N ratio, it is worthwhile to construct rf coils specifically tailored to 57 match the muscle's size and geometry. For these reasons, the experiments in this thesis required construction of NMR probes specifically designed for these studies, which are described below. In situ cat experiments. A Plexiglas®/Lexan® probe was designed and built for these experiments [Figure 6]. One half of a PlexiglaQD tube was used as a cradle to hold the animal. A hollow base along the entire length of the cradle provided a protected area to run the cables for the stimulating wires, force transducer and temperature probe. A 2 cm helmholtz coil was built from 16 gauge copper wire and the rf circuitry [Figure 7] was contained in an aluminum box placed within a holder attached to the sides of the cradle. The circuit could be tuned with the probe inside the magnet. An adjustable knee mount held the joint in place and stabilized the surface temperature probe and platinum. bipolar electrode. A. non—magnetic force transducer was built with four semi—conductor strain gauges (120 ohms, BLH electronics) arranged in Wheatstone bridge configuration [Figure 8], attached to an aluminum cantilever beam. The deflection. of the aluminum. beam changed the resistance in the strain gauges, and the voltage output of the bridge was amplified and recorded (Gouhd Model RS32OO amplifier) outside tflue magnet. The force transducer was mounted on a movable Plexigla§3 base so that the muscle length could be adjusted. An aluminum 58 Figure 6. Plexiglas/Lexan Probe constructed for animal experiments within in a 4.7 Tesla magnet. Force and temperature measurements can be made, stimulating wires allow muscle stimulation, and the helmholtz coil transmits and receives the NMR signal. meet; 6 6 peace 09:1 6 59 0.396.833“ Q \ D p x./ l}[ A n.” ® ® 0 m 0 _ O _ m e / _ ’ Q 0 Legumes: a as @2333 «3.8+ Vice taste mofomE 60 Figure 7. (A) and (B) Schematic of Helmholtz coil and (C) circuitry of the coil. 61 x —:X/2-1 T X(1.1) l Helmholtz Coil C matching capacitor (l-lOpF) \ o—w (im— 7.1 V 'balanccd' tuning capacitors (I-IOPF) Figure 8. Circuit for specially constructed force transducers. 63 owgum 5.3.355 noeuoucz tan—U c.3339: 8:6 emm wowsaw :.auum 64 door for the magnet bore was built and embedded with radio frequency chokes for all wires running through the probe to reduce rf interference from outside the magnet. Isolated perfused muscle studies. A probe previously built in our lab was .modified for the isolated. muscle experiments [Figures 9 and 10]. The probe case was constructed from aluminum. 2% 2 cm helmholtz coil [Figure 7] was nounted.cn1 the top portion (M? the probe with the capacitors directly underneath. The wires of the completed circuit ran ckwni the length (H? the probe tx> a connector. Brass rods were positioned so that the circuit could be tuned with the probe within the magnet. An upper muscle mount was built from DelrinC>(non—magnetic polymer) with a threaded brass rod to allow changes in muscle length. The muscle was emtached tx> specially built force transducers directly' below the Inuscle. The force transducers were built from. four Inicrofoil strain gauges (350 ohms, BLH electronics) arranged in a Wheatstone bridge configuration, [Figure 8] enmi were nmunted (Ni either [@lrinC)(or brass beams. In some experiments a stiff nylon "weedeater" line was tied to the muscle tendon and extended to a commercial force transducer positioned outside of time magnet. All force transducers were calibrated by hanging weights (100 g - 5 kg) and recording the output. A linear response was observed within this range. Two platinum electrodes were 65 Figure 9. Probe for isolated perfused muscle experiments within a 9.2 Tesla magnet. 66 Hemholtz coil ' adjustable muscle mount clelrin muscle clamp muscle coil circuit venous drain 67 Figure 10. Top portion of probe for isolated perfused muscle experiments within a 9.2 Tesla magnet. 68 arterial supply temperature probe /' ”I! a/ bipolar stimulatinq electrode brass heating coil muscle venous drain 69 mounted to allow direct stimulation of'tjuaimuscles at the proximal and distal tendons. A thermistor was placed near the surface (H? the muscle for recording temperature (YSI Model 73A) recorded the surrounding temperature. All wires were connected to radio frequency chokes embedded in an aluminum plate located at the bottom portion of the probe. The remainder' of the cylinder was constructed. to house perfusion lines enui tubing for lflME copper heating coils located directly behind the muscle. Procedures For The Basic 31P—NMR Pulse Experiment The magnetic field strength was either 4.7 Tesla (in situ cat experimentsi or 9.4 Tesla (isolated perfused muscle experiments). In both experiments a helmholtz coil served as both a transmitter and receiver specific for the phosphorus frequency. The phosphorus frequency was dependent on the spectrometer used (81 MHz an: 4.7 Tesla, 162 MHz at 9.4 Tesla). .At the beginning of each experiment, the proton NMR signal (200 MHz at 4.7 Tesla, 400 MHz at 9.4 Tesla) from muscle mater inns monitored while adjusting time magnetic field homogeneity, a procedure known as "shimming". This procedure takes between 10 and 30 minutes, and is important, since if the magnetic field is as homogenous as possible .across time sample, then rim; spectral. peaks are narrowed, and the peak signal to noise (S/N) ratio is 7O correspondingly enhanced. fflme proton signal 16min muscle water was used rather than the phosphorus signal because of its relatively high abundance and sensitivity, which enables high S/N FID's in a single scan. Next, a fully relaxed (15 3 pulse interval, 90° pulse width) spectrum was acquired from resting muscle. Typically 32-64 scan spectra were acquired, requiring 8-16 min. As noted above, these fully relaxed spectra allowed for direct quantitative comparisons of metabolite levels, and (in) be used ti) calculate saturation corrections for other spectra acquired under saturating conditions. For most protocols, the spectrometer's computer was then programmed to acquire a series of from 32-64 spectra, each averaged over 53 15 tx> 60 second tjmme interval. .At the beginning of the third time block, muscle stimulation was initiated. The stimulation was tinimxi off after 2%) min, after time 32nd block was finished. Thus about half the spectra were recorded during recovery after stimulation. Gated 31P—NMR Pulse Experiment Using the above protocol, metabolic events can be monitored with a time resolution of about 15—60 seconds, depending' on time S/N ratio. For‘ some experiments time resolution below 1 s was necessary. Gated NMR can accomplish this time resolution by acquiring each FID signal at precise time points relative tie a contraction, 7l storing the data, allowing the muscle to recover, and then repeating the sequence, while averaging the FID's acquired at each time point (Adams et al., 1990; Foley and Meyer, 1992). The effective tjnme resolution of tinjs method is limited only tn/ the acquisition time of 51 single FID, or typically 140 ms in our experiments. This method was used for the experiments described in Chapter 4, in which the effect of acidosis on the ATP cost of contractions was measured. Data was acquired at specific time points before and following either ea short burst of twitches or 53 single tetanus [Figure 11]. The cycle was repeated several times and time data from each specific: time point was averaged until an adequate S/N ratio was achieved. The difference between metabolite concentrations immediately before and after the contraction yields the changes associated with contraction. MUSCLE PREPARATIONS The advantage of intact tissue is that it allows monitoring of intracellular metabolites continuously in animals and humans (Arnold et al., 1984). Perfused tissue studies allow environmental manipulation by the investigator. Both preparations are limited by tissue heterogeneity and the fact that the signal is averaged over cell populations and time. It is important to assess organ Figure 11. Gated 31P-NMR Experimental protocol. Pulses are given at each time point, the first given immediately after contraction . The signal is temporarily stored and after several repetitions, signals at each time point are averaged. 73 GATED NMR PROTOCOL |.O kg —— A I I i i i l l , , I I I I 7; ” l ” l ,1 (i 0.0 2-0 2-5 l5.0 30.0 60.0 '2 -0 Time (seconds) T - rod/o frequency pa/ses A = spec/rome/er fr/gyers [some/Ho con/roo/fon 74 metabolic stability, in addition to assessment of metabolite levels obtained by NMR (Balaban, 1984). In Situ Soleus Studies Surgical techniques. Cats (4—5 kg) were anesthetized with ketamine chloride (15 mg/kg) subcutaneously, and sodium pentabarbital (30 mg/kg) intravenously. The carotid artery was cannulated to monitor arterial pressure and obtain blood samples. The animal was ventilated and hemodynamically stable throughout the experiments. Rectal temperature was maintained at 37°C by a heating pad placed underneath the animal. Supplemental fluids and anethestic were given intravenously as needed during the entire experiment. Tflme calf muscle group (gastrocnemius, plantaris and soleus) was exposed by dissecting away the skin and outer fascia. All tendons were chssected from time calcaneus. The soleus tendon VWMS dissected free from Igastrocnemius tendons enmi ligated. The muscles surrounding time soleus were carefully reflected away. The femoral artery branch that supplied the soleus also had sub-branches that supplied the gastrocnemius and several anterior compartment muscles. The branches rmfl: directly supplying time soleus muscle were double ligated and severed. The venous branches paralleled time arterial configuration eumi were also ligated. and cut. The proximal insertion remained 7S intact and the muscle was perfused by the animal's circulation. The proximal joint was stabilized by a tungsten pin inserted in the bone and fixed to a Lexan® support. The distal tendon was attached to a force transducer. The soleus motor nerve was isolated, severed distally, and placed in the bipolar platinum electrode for muscle stimulation. Muscle temperature was monitored by a surface probe placed on the exterior surface of the muscle and maintained within a physiological range (35°-37W3). The muscle was bathed in mineral oil and wrapped in Parafilnm). The muscle was stretched to optimal length and maximally stimulated (S-IOV, l rm; duration) with 51 Grass stimulator generating isometric twitches. Taken together, these procedures required around 5 hours from the time the animal was anesthetized until the first spectrum was acquired. Additional surgery was required for oxygen consumption experiments. The venous branch that normally drains the blood supply of anterior compartment muscles, was ligated and «cannulated (PE SN) tubing) for 'blood sampling. The proximal femoral venous branch was clamped during sampling so blood flowing from the soleus muscle could be collected in an air tight Hamilton®> (Hospex) syringe from the cannula. Arterial blood samples were taken from the carotid artery before and after each stimulation period. 76 Isolated Perfused Muscle Studies Isolated perfused cat soleus and biceps muscles preparations (Kushmerick anmi Meyer, 1985; iMeyer et al.,1985) enable study CH? the contractile and Inetabolic properties of muscle contraction with greater control ofexperimental parameters, at the cost of considerably greater surgical preparation. Cat biceps (>70% fast glycolytic, >25% fast—oxidative glycolytic) and soleus (>95% slow oxidative fibers) muscles are relatively homogenous in cell fiber type thus reducing the complication (Hf energetic parameters being attributed to different fiber types within the muscle rather than metabolic changes within the myocyte. Surgical procedures. Cats (4—5 kg) were anesthetized with ketamine chloride (15 mg/kg) subcutaneously, and sodium pentabarbital (30 mg/kg) intravenously in all experiments. The animal was ventilated throughout the surgery. Rectal temperature was maintained an: 37%: by a heating Emmi placed underneath time animal. Supplemental fluids and anethestic were given intravenously as needed during' muscle isolation. of either the soleus or biceps muscle. Soleus Isolation: Surgical procedures were similar to those previously described (see Insitu Muscle Preparations section) with some modifications. mee calf muscle group was exposed and the soleus tendon was dissected free from 77 the calcaneus and all other tendons and ligated with fishing line. The muscles surrounding the soleus were carefully reflected away. All arterial and venous branches not supplying or draining the soleus were double ligated and severed. The femoral artery was isolated anteriorly to allow cannulation with IT: SO tubing. The \mnii was also isolated and cannulated with PE 90 tubing only in experiments in which oxygen consumption was measured. The soleus nerve was cut and cauterized. The muscle was entirely isolated, except for its proximal insertion on the tibia. This bone was cut anmi the soleus muscle was removed from the animal. Braided, nylon fishing line (25 lb. test) vmms secured around the kxnme and used ti) attach the muscle to the upper muscle mount. In this case, the entire procedure, from. anesthetizing the animal to acquisition of the first spectrum, typically took 6-8 hours. Biceps Isolation: The forelinm> muscle groups were exposed by removal of skin and outer fascial Several muscles comprised a layer covering the biceps muscle and were cauterized and reflected away. The distal tendon was isolated and severed from its radial insertion. The tendon was ligated with fishing line. The fascia surrounding the biceps vmms carefully {MENU} away exposing time surrounding vessels. The biceps is; supplied eumi drained tux several arterial and venous branches located primarily on the 78 proximal and distal portions of the muscle. These branches stem from an artery and vein running parallel to the muscle. All branches not supplying the biceps were double ligated and severed. All nerves were cut and cauterized. The muscle was isolated, and the proximal tendon insertion was severed iflimi the bone emmi also ligated vniii fishing line. The proximal tendon was attached to the upper muscle mount. Both muscles (soleus and biceps) were bathed in mineral (NJ. and surrounded by 21 plastic tube Kid transparency film PP2200). The distal tendon was attached to a force transducer. Platinum electrodes were place on the proximal and distal portions of the muscle for stimulation. Temperature was monitored by a surface probe placed on the exterior surface of the muscle and maintained within a physiological range (35-37°C). The muscle was stretched to optimal length and maximally stimulated (10- SOV, 1 ms duration) with a Grass stimulator. Perfusion solution. Kreb‘s Heneleit solutions (KHS) were prepared for blood washing and final perfusion solutions a day prior to the NMR experiments (storage 4°C). The final perfusion solutmmi was prepared the ckny of the experiment. KHS Preparation: Solutions were prepared for use at either 37°C or 20°C for perfusion solutions or blood washing, respectively. The solutions contained NaCl (116 79 mM), KCl (4.6 mM), KH2P04 (1.16 mM), NaHCO3 (24 mM), CaCl2'2H20 (2.5 mM), MgSO4°7H20 (1.16 mM), to mimic physiological conditions enmi gentamycin sulfate KN) mg/L) to prevent bacterial growth. .All solutions were bubbled with 95% CD enmi 5% C02 and filtered (Fisher 0.2 um clay filters). For KHS (37°C) glucose (5 mM) and sodium pyruvate (0.15 rmn were added ti) provide substrates, as well as papaverine HCl (10 mg/L) to prevent vasoconstriction.. Phenylphosphonate (PPA) was brought to physiological pH with sodium hydroxide and added to the KHS (37°C) solution (PPA 10—15 mM final concentration). PPA is used for sue extracellular volume and gil marker, since it has been shown not to cross the muscle cell membrane, and its NMR chemical shift is sensitive to pH within the physiological range (Kushmerick et al., 1982). Red Blood cell Ekeparation (Blood hashing): Packed sheep :red tflmmmi cells (400 Hflq Lampirez Biological Labs) were divided evenly into six 250 rml polycarbonate centrifuge bottles. The bottles were then filled with 0.9% filtered saline solution for rinsing. The cells were then spun an: 1800 RPM (refrigerated centrifuge) tin: 10 minutes at 10°C and the supernatant aspirated. This procedure was repeated 3 times with saline solution and 3 times with KHS (20°C). Perfusion Solution Preparation: The final perfusate consisted of filtered 3.5% bovine serum albumin, 12 — 15% 8O washed red blood cells and 856 KHS (37°) (Meyer et al., 1985). Perfusate was mixed every hour in 60 ml batches of filtered KHS solution and albumin, and washed erythrocytes. Perfusion Set-Up: The perfusate was gently stirred in a round bottom beaker and peristaltically pumped at 0.2 — 0.4 ml/min/g muscle through a brass artificial lung, a bubble trap, and then to the muscle [Figure 12]. Silastic <3 tubing was used throughout the lung to allow gas diffusion. All remaining tubing was TygonC>(gas impermeable) to prevent gas equilibration with room air. r a 81 Figure 12. Isolated perfused muscle experimental set up. EoLp w=OCw> Lo LOUoc cmpc_wxo p we coospmcoc_ otawmocm no mu no we: _D_L®T:U co_.o_:uc_ «to; mc__dEOm mote m_nrsn Locc__w o 0_Uw:E mesa o_. o.m_coa t_0>cmmmc c.0wsetma omega LE: CHAPTER 3 CONTROL OF RESPIRATION BY PHOSPHORYLATION STATE IN SLOW-TWITCH MUSCLE INSITU. INTRODUCTION There is a balance between energy production and utilization in skeletal muscle cells indicating metabolic control. ATP and PCr provide chemical potential energy for energy requiring processes (Kushmerick, 1983). The primary source of ATP at rest and during mild stimulation is oxidative phosphorylation. There is considerable controversy over the exact signal which links the rate of oxidative phosphorylation to the rate of ATP utilization in muscle. The major candidates for this control are phosphorylation state (relative concentrations (fl? adenine nucleotides, phosphocreatine, and Pi), redox state (ratio of NADH and NAD+), and oxygen delivery (Balaban, 1990). Metabolic control may be different in distinct skeletal muscle types, since the different fiber types have diverse metabolic characteristics. Slow—twitch fibers have a higher mitochondrial content, lower glycolytic capacity and lower actomyosin activity than fast-twitch fibers, as well as lower resting levels of PCr and ATP and higher Pi (Lawrie, 1953; Meyer 6%: al., 1985). The time course for 84 PCr changes is; faster anmi oxygen consumption is; lower at identical twitch rates in slow—twitch fibers than in fast- twitch fibers. 0n the other hand, mitochondrial structure is similar between muscle types, and although slow-twitch muscles have 63 higher aerobic capacity' than fast-twitch muscles, when corrected for mitochondrial content, the values of maximal oxygen consumption are equivalent (Hoppeler et al., 1987). Most studies of respiratory control in mammalian muscle have been conducted on human muscles of mixed fiber type, or on animal muscles with predominantly fast-twitch fibers. These studies almost unanimously agree that changes in phosphorylation state can account for changes in respiration, although there is still disagreement on whether this dependence is due to control of respiration by ADP, or by phosphorylation potential (Foley et al., 1991; Meyer, 1988; Meyer, 1989; Meyer et al., 1986; Meyer et al., 1985; Kushmerick.eet al., 1992; Nioka (at al., 1992). In contrast, studies of heart muscle indicate that changes in respiratory rate can occur with no change in phosphorylation state (From. et al., 1986; Katz et al., 1987; Hassinen, 1986; Balaban, 1990; Heineman and Balaban, 1990; Katz et al., 1988). This indicates that some other controlling mechanism, possibly redox potential, must be at play in the heart. Mammalian slow-twitch..musclei has characteristics of both heart and fast—twitch muscles from the metabolic point 85 of view. Like cardiac muscle, slow-twitch muscle is almost totally dependent on aerobic metabolism for ATP production, and contraction is rapidly reduced if blood flow or oxygen supply is efldndnated (Meyer and Terjung, 1980). On the other hand, like other skeletal muscles, it is not spontaneously active, can be tetanized, and is not sensitive to extracellular calcium changes (Crow and Kushmerick, 1982). hi view of this, it seems reasonable that if there is a mechanism for respiratory control independent of phosphorylation state in heart muscle, this mechanism should be evident to some extent in slow-twitch muscle. A recently published study' of perfused cat soleus muscle suggested control of respiration in slowwtwitch muscle vmms similar ii) that iii heart (Kushmerick (at al., 1992). Although there was a roughly linear relationship between PCr and oxygen consumption at relatively low stimulation rates, as stimulation rate was increased, steady—state PCr changed less dramatically. Furthermore, during recovery after stimulation, PCr recovered to values above those observed before stimulation. These observations are rmn: consistent with control kn/ a single factor such as phosphorylation state. The purpose of this study was to examine the relationship between phosphorylation state and oxygen consumption in cat soleus musche in situ. One series of experiments monitored Twin ATP, EM. and til noninvasively 86 during submaximal stimulation in intact soleus muscles using 31P—NMR. Another series measured oxygen consumption during steady-state conditions at identical stimulation rates. The results do rmfl: confinn the previous study of perfused cat soleus muscle, and suggest that phosphorylation state is; the dominant regulator of respiration in slow-, as well as fast-twitch muscle. MATERIALS AND ME THODS Surgical Techniques Cats (4—5 kg) were anesthetized with ketamine chloride (15 mg/kg) subcutaneously, and sodium. pentabarbital (30 mg/kg) intravenously. The carotid artery was cannulated to monitor arterial pressure and obtain blood samples. The animal was ventilated and hemodynamically stable throughout the experiments. Mean arterial blood pressure was 115 i4 mmHg (mean iSE, n=12). Rectal temperature was maintained at 37°C vnlii a heating Emmi placed underneath time animal. Supplemental fluids and anesthetic were given intravenously as needed. The soleus muscle was isolated as described previously (see Chapter 2). In brief, muscle groups surrounding the soleus were gently reflected away. The soleus tendon was dissected free from the calcaneus and all other tendons and ligated. The soleus blood circulation was isolated. The branches not directly supplying or draining the soleus 87 muscle were double ligated and severed. The proximal insertion remained intact and the muscle was perfused by the animal's circulation. The proximal jcflim: was stabilized by a tungsten pin inserted in the bone and fixed to a LexamO support. The distal tendon was attached to a force transducer. The soleus Hmtor Imnmme was isolated, ligated, and placed in a bipolar platinum electrode for muscle stimulation. Muscle temperature was monitored by a thermistor placed on the exterior surface of the muscle and maintained lNlthln El physiological range (35°—37W3). The muscle was stretched to optimal length and maximally stimulated (5—10V, 1 ms duration) with a GraseO stimulator at 0.5, 1, 2L 3, and (4 Hz, (order randomized) for 15 minutes. These stimulation. rates ‘were selected. because preliminary experiments indicamad that they maintained a metabolic and mechanical steady—state. Experimental Series I/Phosphorous NMR Cats (nz6) were placed in a Eflexiglan/Lexamo pmobe constructed for this project [Figure 6]. The soleus muscle was positioned within iriaa 2 cm diameter Helmholtz coil. The tendon was attached to a non~magnetic force transducer for isometric force measurements. The Imiime was placed within a GE Omega 4.7 Tesla magnet. The circuit was tuned to 81 MHz and the magnetic field shimmed on protons of muscle water. Control spectra were acquired (pulse width = 90°, rd = 15 s, ns = 8) at the beginning of each experiment 88 and between stimulation bouts. Sixty—two phosphorus spectra (pulse width == 60°, rd == 1.87 EL ns == 8) were continuously acquired during stimulation and recovery. The muscle recovered completely between stimulation bouts, as indicated by restoration of phosphate metabolites and force to control levels. FID's were zero-filled to ¢M< data points and multiplied tux an exponential corresponding to Iii Hz line broadening. Peak integrals were computed after baseline correcthmn and Fourier transformation. Steady-state PCr levels were determined by averaging the PCr integral of 20 spectra acquired during the final 10 minutes of stimulation and by calculating a percentage from control spectrum immediately prior to stimulation. Percentage values were multiplied by 12.7 moles PCr/gram wet weight muscle (Meyer et al., 1985). Exponential time constants for PCr changes were computed by non-linear least squares fit. Intracellular pH was estimated from the chemical shift of the inorganic phosphate peak (Moon and Richards, 1973). Experimental Series II/Oxygen Consumption In addition to the surgical techniques described above cats (n=6) underwent the following surgical procedures. The femoral venous branch, that normally drains the blood supply (If anterior compartment .muscles, was ligated. and cannulated for blood sampling. Arterial blood samples were 89 taken from the carotid artery before and after each stimulation period. The distal tendon was connected to a commercial force transducer (Grass instruments). For each stimulation period (same frequencies and duration as above) three successive venous blood samples were taken during the final ll) minutes (fl? stimulation. This vmms during' the metabolic and mechanical steady-state as determined by PCr levels and force measurements of 31P-NMR experiments. Blood. flow Ineasurements were taken tn/ collecting 'venous effluent for 15 seconds. Blood oxygen content was measured by a LexO2Con Oxygen Analyzer (Lexington Instruments). Oxygen consumption was calculated by multiplying (arterial - venous) oxygen content differences by iblood flow and dividing by the muscle wet weight. Results Figure 13 shows a series of 31P-NMR spectra for a cat soleus muscle in situ during 15 minutes of stimulation (2 Hz) and recovery. Each spectrum is the smmi0.05). ATP levels were constant during stimulation and recovery. Figure 14 shows the mean PCr changes at stimulation rates of 0.5, 1, 2, 3, l-) 90 FIGURE 13. Series of spectra of soleus muscle, control (1 minute), during 3 Hz stimulation (15 minutes), and during recovery (15 minutes). Each spectrum is an average of 16 scans (60 pulse width, rd = 1.62 s). 9| PCr ATP Figure 14. PCr levels from 31P-NMR spectra of cat soleus muscle acquired during and after 15 minutes of stimulation at 0.5, 1, 2, 3, and 4 Hz. Exponential lines were computed assuming a single time constant (0.83 min) and 95% recovery of signal intensity. ‘3 3O i.el. aIxfllf Iii: To i % alTQIlo To Zeal. 014;: alIIoll Z . c __ 8 _o .utfillllo H Textile O 4 . Lou .[xo 2 Th 00155.. A ll». lo Al .léwl 0 Z all H O A d l 5 3 Tillie o A i all 0 0 l o Lire 01 A Z S} ’ 2 zl. ol 0 o e i l $01 01 a 0 Z lo all oll Xi H lo 1 ol 1. o l A 0 ol ). A oli i To Ali 0. .l n i All. TI Ten i o .. 0 i 5 To 2 o lo A Z To l o .lo A H all 0 ol ill. 5 o n o .lo l o .l 01 ll 0 all _ A. — q _ u _r _ w 0 5 O 5 O 5 O 5 O 5 O 5 O 5 O O O 9 9 OO 00 7 7 6 6 5 5 4 Ar All Ali MINUTES 94 TABLE 2 PCr time constants Onset stimulation 0.85 i 0.8 Recovery 0.83 i 0.1 (Values are means i SE given in minutes; onset n=16, recovery n=17). r“ 95 and 4 Hz. Steady-state PCr levels (mean over last 10 min) decreased with increases in stimulation rate. The overall mean time constant for PCr changes was 0.83 i 0.07 minutes [Table 2]. The time constant of PCr changes was independent of stimulation rate, as sflmwni in Figure 15, gum) similar at onset of stimulation versus during recovery [Table 2]. Table 3 summarizes intracellular pH, oxygen consumption and twitch force data at rest and in the steady-state during repetitive stimulation. Resting oxygen consumption was 0.185 t 0.04 umol 02/min/g and blood flow was 0.075 i .007 ml/min/g at 37°C [Table 3], comparable to previous studies <)f intact (Bockman, 1983) anmi perfused soleus muscles (Meyer et al., 1985). Intracellular pH was not different from control values during steady—state stimulation. Steady-state rmmfl: force (decreased vniii an increase in stimulation rate. Oxygen consumption and PCr levels were proportional to the product of peak force and stimulation rate [Figure 16, 17]. Steady' state oxygen consumption was a linear function of steady-state PCr levels, (r = 0.965) with slope = —0.15857, and y - intercept = 3.432 [Figure 18]. 96 Figure 15. PCr time constants for stimulation rates of 0.5, 1, 2, 3, and 4 Hz. PCr time constont (min) 97 4 T 3 l 2 —4L— 0 <> 0 o O O O O l ‘” n n g o 9 o o o 0 O . 8 8 § O 1 1 l 1 1 1 J O.5OO i.OOO 1.5OO 2.000 2.50O 3.000 3.5OO 4.000 Stimulotion Rote (HZ) 98 TAB LE 3 Hz 0.0 0.5 1 2 3 4 pH 7.05 7.07 7.09 7.05 7.08 7.09 i0.04 i0.02 i0.05 i0.04 i0.02 i0.03 Force 94 95 95 87 79 %control i2.0 i2.8 i4.5 $2.6 i2.0 V02 0.185 0.365 0.567 0.780 1.11 1.15 umol/min/g i0.04 i0.07 i0.10 i0.16 i0.10 i0.09 Blood Flow 0.075 0.930 0.114 0.121 0.160 0.160 ml/min/g $0.007 i0.015 i0.021 i0.025 i0.027 i.033 (Values are means i SE.) 99 Figure 16. Steady-state oxygen consumption vs. product of stimulation rate times mean peak twitch force (percent of initial i.e., first twitch of each stimulation series) during soleus muscle stimulation (r = 0.98). \702 (umol Oz/g muscle/min) 1.50qu 1400- $300- .ZOO— .iOO~ .OOO— Cl900— (lBOOe (l700— (l600~ (JSOO- (l400~ (JBOO~ (l200i l l l l 0100—1 (lOOO 100 l J l l l l l j l 50 100 150 200 250 300 35G Force >< Rote (% control/sec) l0] Figure 17. Steady-state PCr vs. product of stimulation rate times mean peak twitch force (percent of initial i.e., first twitch of each stimulation series) during soleus muscle stimulation (r = 0.96). PCr (% initiol) L I 5O 1 l l l l l 100 15O ZOO 25O BOO 350 Force X Rote (% control/sec) Figure 18. Relationship between steady—state PCr and oxygen consumption at rest and during stimulation (0.5, 1, 2, 3, 4 Hz). PCr levels are from figure 15, assuming an initial PCr value of 12.7 mol/g muscle (Meyer et al., 1985). Values for oxygen consumption are from Table 3 (Slope = —0.15857, y intercept = 3.432, r = 0.97). l04 @— l-O-l o_om3E0\o_oE3 00o 0e —1 v. 000.0 10070 I00m0 (000.0 -l00¢.0 1000.0 I000.0 #0050 [000.0 (000.0 1000. -007 1-00m. 1-00m. (00¢. I 000. l l l \—V—‘—‘_'-\_"— (quu/alosnuu 6/30 |OL1JI1> ZQ /\ l05 DISCUSSION Our study shows oxygen consumption is a linear function of steady-state PCr, for moderate ATPase rates of slow-twitch muscle in situ. These results are not consistent with a previous study of isolated perfused slow- twitch nmscle, :hi which respiration rate vmms observed to become less dependent on steady-state PCr at higher ATPase rates (Kushmerick 6%: al., 1992). The difference nmu/ be because blood flow became limited at the higher stimulation rates ii) that study. Limited blood iflinv might result iii increased lactate/pyruvate ratio, and hence increased redox potential, which could persist during the recovery period, and drive PCr to a higher level compared to before stimulation. Alternatively, the liwmn: temperature (30°C) at which that study was conducted may also contribute to the difference between results. In any case, our results suggests that changes LUl phosphorylation state are sufficient ti) explain respiratoryI control iii slow-twitch muscle. However, our results (i) not distinguiSh between control lug ADP versus by phosphorylation potential (ln[ATP]/[ADP][Pi]). There was a tmdance between power output and energy production, which depended both on respiratory control, and on the control of twitch force. Oxygen consumption values did not increase linearly with stimulation but were proportional to steady—state force times stimulation rate. Yet, steady-state isometric twitch force decreased as 106 twitch rate increased. This decrease is not analogous to the fatigue which is observed during more intense or ischemic stimulation, because ii: was not associated with acidosis, and because, a steady—state of twitch force was rapidly achieved, and then maintained for many minutes of stimulation. These observations suggest that there is ea rate dependent mechanism modulating calcium release during twitch stimulation of slow—twitch muscle. 'The details of this mechanism are obscure, but deserve further investigation. Near—equilibrium thermodynandc: models; of control of respiration predict that oxidative rate should be dependent on the chference between the cytoplasmic free energy of ATP hydrolysis and an intramitochondrial energy term related ti) redox potential (Erecinska anmi Wilson, 1982). If the redox potential remains constant, respiration rate should depend linearly on cytosolic phoshorylation potential over a) submaximal range CM? respiratory rates. This basic hypothesis has been incorporated into an electrical analog model of the relation between PCr changes and respiration in skeletal muscle (Meyer, 1988) This model predicts that steady-state respiration should be linearly dependent on PCr levels during stimulation at rates within the maximum aerobic capacity of time muscle. The slope of this steady state relation is dQOZ/d[PCr] = - 1/(pt), with p 2 P/Og, and T = apparent time constant for PCr changes. The model also predicts that the time 107 constant for PCr changes at the onset of stimulation and during recovery should be the same, and both should be independent (Hf stimulation rate. Also, these PCr time constants should kme relatively Shorter iii muscles with higher ndtochondrial content and/or lower total cmeatine levels. The results of this study are basically consistent with time predictions (Hf this model. First, steady-state oxygen consumption. was 23 linear functicmi of PCr levels [Figure 18]. The P/02 ratio 1%“; estimated to kme 7.9 by applying the equation above using the slope of the relationship between steady-state PCr levels and. oxygen consumption (Figure 18) and the mean time constant for PCr changes (0.83 min). This value is reasonably close to the expected value CH’ 6 (Lemasters, 1984). Second, time PCr time constant was independent of stimulation rate, and not significantly different between time onset (If stimulation and during recovery. Thind, as expected from time higher mitochondrial content and the lower total creatine content of slow—twitch. compared U) fast-twitch Inuscle, time mean time constant (0.83 min) was less than that observed in rat fast-twitch muscle (1.4 min) (Kushmerick and Meyer, 1985; Foley et al., 1991). Thus, these results are consistent with the liimmnr model, and therefore enme consistent Math respiraticmi rate tinitrolled tm/ phosphorylaticmi potential. However, ems pointed (mm: by Connett, these results (i) not rule out metabolic control by ADP availability. 108 In summary, we found no evidence for ee non— phosphorylation state mechanism for respiratory control in cat soleus muscle in sniir We conclude that respiratory control in slow—twitch muscle is fundamentally the same as in fast—twitch muscle being primarily controlled by phosphorylation state. 109 CHAPTER 4 EFFECT OF HYPERCAPNIC ACIDOSIS ON THE ATP COST OF CONTRACTIONS IN FAST— AND SLOW-TWITCH MUSCLES INTRODUCTION Although there is general agreement that respiration is controlled by cytoplasmic phosphorylation state within maximum aerobic capacity in skeletal muscle, the specific controlling parameter is INN: clear (Mahlery 1985; Meyer, 1989; Meyer et al., 1986; Chance et al., 1986). The two most likely7 candidates are (cytoplasnur: AIW’ concentration and cytoplasmic phosphorylation potential. These two hypotheses are difficult to distinguish by simple correlative experiments because both ZUH> and cytoplasmic phosphorylaticmi potential change iii tandefli during inuscle stimulation. Connett has shown that study of contracting muscles during experimentally induced acidosis could, in principle, distinguish between control by ADP versus phosphorylation potential (Connett, 1988a; Connett, 1988b). However, a potential obstacle to the design of such a study is the possibility that acidosis alters the utilization of ATP, as vmetl as ATP synthesis. For example, ii? acidosis profoundly inhibits cross—bridge cycling, it would be difficult to increase respiratory rate by muscle stimulation in acidic muscles. H0 Acidosis has long been reputed to have a significant role in muscle fatigue (Curtin et al., 1988; Lannergren and Westerblad, 1989; Dawson et al., 1980; Chance et al., 1985; Miller et al., 1988; Westerblad and Lannergren, 1988). Fatigue is the decline of force observed during repeated contraction of skeletal muscle at power outputs above those which can be maintained by aerobic metabolism. It is well known that fatigue is associated with decreased ATP utilization (Close, 1972; Dawson et al., 1978; Terjung et al., 1985; Dawson et al., 1980). Skinned muscle fiber studies suggest that decreased intracellular pH is an important cause (n? muscle fatigue (Cooke EN: al., 1988). Lowered pH decreased the calcium sensitivity of the contractile apparatus, (Fabiato and Fabiato, 1978; Donaldson and Hermansen, 1978), the peak force during maximum calcium stimulation, and the iiuie of cross-bridge cycling and shortening velocity (Chase and Kushmerick, 1992; Cooke et al., 1988; Godt and Nosek, 1989; Metzger and Moss, 1987). If these effects occurred iii intact muscle (Hultman et al., 1985), the result could be profound fatigue and a decrease in ATP use during contraction under acidic contractions. On time other hand, studies (Hf intact muscles Ci) not consistently show a good correlation between acidosis and fatigue. In particular, Adams (1991) found little effect of hypercapnic acidosis on peak isometric tetanic force in cat skeletal muscles, although peak twitch force and the 1H rates CM? force (development. and .relaxaticmi were reduced. Unfortunately, these results do not prove that ATP utilization is not sensitive to acidosis, since the actual rate of cross bridge cycling or shortening velocity was not measured. Thus, it is possible that the economy of isometric force development is increased by acidosis, so that less ATP is required to maintain isometric force. The economy of muscle contraction, the relationship between isometric mechanical response gum) energetic cost, varies among animals as well as between fiber types. The frog sartorius 'muscle is (M) times less economical than tortoise skeletal muscle and 100 times less economical than mammalian smooth muscle. The energy cost cflfzmouse fast- twitch muscle was measured to be from 50—300% greater than slow—twitch muscle depending on duration of the stimulation (Crow and Kushmerick, 1982; Close, 1972; Rall, 1972). There are several chemical reactions that contribute to the energy cost of muscle contraction (Rall, 1972; Kushmerick, 1983; Kushmerick et al., 1969; Bienfait et al., 1975). Energy release during steady—state contraction is primarily determined by actomyosin ATPase (70%) at the crossbridges, and Cir?“ ATPase (30%) EN: the sarcoplasmic reticulunn There is a) direct relationship between actomyosin ATPase rate and maximum velocity of shortening (Nakamaru.enmi Schwartz, 1972). .A change iii hydrogen ion concentration may alter ATPase activity and affect the energy cost of contraction. H7 The purpose of this study was to directly measure the effect of tumaicapnic acidosis (n1 ATP utilization during isometric contractions of perfused cat fast— and slow— twitch muscles. This information was a prerequisite to the design of the following study (Chapter 5), since that study required selection ci'ee range of stimulation rates which would result :Ui similar rates (Hf ATP utilization during normocapnia and hypercapnia. ATP utilization was observed during acidosis with gated 31P—NMR (Foley and Meyer, 1992; Adams 6%: al., 1990) iii isolaUai cats soleus enmi biceps Hmscles. The results show that the ZVH? cost of tetanic contractions is reduced in proportion to the reduction in force. Thus, the intrinsic rate CH? cross—bridge cycling and. the economy7 of force development appear‘ to not Ibe sensitive to changes in pH over the range studied (ph 6.6 - 7.1). MATERIAL AND METHODS Surgical Techniques Cats were anesthetized vniii ketamine subcutaneously and pentobarbital intravenously. 21 tracheotomy was performed to allow controlled ventilation. Either the soleus or biceps muscle was vascularly isolated and excised, as previously described (Chapter 2). The artery supplying the muscle was cannulated and perfused at 0.2-0.4 ml min‘1 g’1 (perfusion pressure 80 — 100 Torr) with a 15% H3 suspension of sheep red blood cells in bicarbonate-buffered Krebs-Henseleit solution. Perfusate was equilibrated with either 5% CO2—95% 02 (normocapnia) or 70% C02-30% 02 (hypercapnia) at 37°C in a SilastidO tube oxygenator. The isolated muscle was pdaced within a 2? 0n Helmholtz coil mounted. in ee custom) made 7.4 cm) diameter" probe. Both tendons were secured by custom built DelrinC> clamps. Platinum wire electrodes were securely placed on each end of time muscle and isometric force recorded ems described previously. Supramaximal isometric twitch contractions of soleus (1 Hz, 10 s) and biceps (2 Hz, 10 s) were induced by a square wave pulse of a Grass stimulator (20—50 V). Tetanic contractions of soleus and biceps were given at 30 Hz for 2? s and iHM) Hz for l. s, respectively. Each stimulatiCM1 protocrd. was administered 6N; normocapnia. and during hypercapnia. Gated 31P-NMR Experiments Spectra of isolated perfused muscles were acquired at 162 MHz on a Bruker AM400 wide bore spectrometer. Control spectra (90° pulse, 15) s interval, ns == 1, 4, E3 or 16 scans) were acquired prior to each stimulation series and during the time interval of gas equilibration. The gated protocol (see Chapter 3) was implemented, with the first scan acquired 0.5 s after the end of the tetanus or burst of twitches, and with 6 successive scans following at H4 intervals of 15, 30, 45, 60, 120, and 240 EL The 240 s scan served and the pre—contraction spectrum. FID's were zero-filled to IH< data points and multiplied by an exponential corresponding to 15-25 Hz line broadening before Fourier transformation. Control spectra were integrated and PCr/ATP ratios were determined during normal and low pH. Spectrum 7 (240 5) represented control levels of phosphates and was compared directly to spectrum I acquired (LE) 5 immediately following contraction. The percent decrease in PCr was calculated and converted to u mol ATP/g muscle by multiplying the ratio of PCr/ATP in the control spectra, and by the content of ATP measured chemically in previous studies (5.03 umol/g for soleus, 8.9 umol/g biceps, Meyer 6N: al, 1985). Intracellular til was estimated from the chemical shift of the inorganic phosphate peak (Moon and Richards, 1973). Extracellular pH was determined from the chemical shift of the PPA peak (Meyer et al., 1985). RESULTS Table 4 shows the extracellular and intracellular pH, PCr/ATP ratios, anmi contractile characteristics iii both muscles during normocapnic and hypercapnic perfusion. The results are basically the same as reported previously (Adams et al. 1991). Intracellular pH decreased from 7.2 to 6.6 in both muscles. There was no significant decrease H5 in PCr/ATP ratios in either muscle during hypercapnia as compared ti) normocapnia. There 1mm; also rm) significant effect of hypercapnia on peak tetanic force, but peak twitch force decreased by about half in both the soleus and biceps muscles. There was a significant increase in relaxation time after tetanic contractions in both muscles, and a slower rise time in the soleus muscle only with acidosis. Representative gated phosphorus spectra are shown in Figures 1E) and 20 :U1 soleus anmi biceps muscles, respectively, before gum) after isometric contractions, during normocapnia and hypercapnia. Figure 21 summarizes the calculated change in PCr associated with twitch and tetanic contractions in the two muscle types under both pH conditions. There was a reduction in PCr levels and corresponding increase in EH. during" contraction iii both muscles. There was no significant difference in PCr changes associated with the tetanic contractions in either muscle type. (hi the other hand, there was a) significant decrease in PCr cost of twitch contractions in the biceps, and a similar trend was evident in the soleus. Thus, the effects of acidosis are roughly proportional to the effects on isometric force in both muscle types [Figure 22]. H6 TABLE 4 BICEPS SOLEUS normo- hyper- normo- hyper- capnia capnia capnia capnia [PCr]/[ATP] 3.55 3.64 2.94 3.0 i0.51 i0.58 i0.49Ii.——Iii i0.60(n=12) pHiC 7.19 6.56 7.15 6.57 i0.02 i0.05* i0.06 i0.06* pHec 7 52 7 74 7.51 6.78 i0.03 i0.04* i0.04 i0.07* TWITCH rise time ms 28 26 103 102 :3 i1 i10 i10 relax time ms 23 22 124 195 i4 i2 i6 i30 peak force g/g 188 99 93.2 48.7 i33 i14‘ i12 i15* TETANIC rise time ms 82 114 380 530 i5 i10* i29 i56* relax time ms 37i 90 129 259 5 i14* i14 i40* peak force g/g 738 620 323 270 i96 i82 i39 i36 (Values are means i SE, n=7 unless noted otherwise. *Significant difference between normocapnia and hypercapnia by paired Student's t—test p<0.05). H7 Figure 19. Spectra acquired at rest and following isometric twitch and tetanic contractions in soleus muscle during normocapnia (pH = 7.2) and hypercapnia (pH = 6.6). (A) twitch, pH = 7.2, (B) twitch, ph = 6.6, (C) tetanus, pH = 7.2, (D) tetanus, pH = 6.6. immediately 118 A B SOLEUS SOLEUS REI'F 3.0 P?! 5 HT -_ é ' H B 7 o 1 TWITCH P TWITCH PH = 6.5 - 1 l l l. I ll 11 )1 ' were“ 1%" U __h¥ -—_ :0 < ' .1. g 0 "a REST C SOLEUS SOLEUS REST ‘ ‘_¢ v—ih "n 5 1—77 13 gwn 5 P“ n 7 . 1 TETANUS TETANUS MAW 0.: é ' ‘ "n I 6 ‘° rrn H9 Figure 20. Spectra acquired at rest and immediately following isometric twitch and tetanic contractions in biceps muscle during normocapnia (pH = 7.1) and hypercapnia (pH = 6.5). (A) twitch, pH = 7.1, (B) twitch, pH = 6.5, (C) tetanus, pH = 7.1, (D) tetanus, pH = 6.5. BICEPS - PH ‘ 7-1 REST -__ h rn TWITCH lo ____~___________fi r" C BICEPS REST m A} ' "n pH - 7.1 TETANUS _.___' - y lo 5 PPH B BICEPS REST TWITCH 1) BICEPS REST TETANUS pH - 6.5 IO (Ii-t d Figure 21. Energy cost (umol PCr/g muscle) per isometric contraction (tetanus or 10 twitches) during normocapnic (5% C02-95% O2) and hypercapnic (70% CO2—30% O2) perfusion of soleus and biceps muscles. 122 101 [:1596 002 Tetanus -70% C02 1 sec. 3 _ 1 5 10 Twitches 1 e 5 - \ g \ 3 sec. 3 S + A O S [7 a h E Biceps Soleus Biceps Soleus Figure 22. Energy cost Umfl. PCr/g muscle) and force production per isometric contraction (tetanus or 10 twitches) during normocapnic (5% CO2—95% 02) and hypercapnic (70% CO2—30% O2) perfusion of soleus and biceps muscles. 124 alosnui 6/93104 5 320m 385 320m 302m 0 x i A / . / e -E L H - % 00—. it h - H - h H com -. W 1, 00m) 11 .OQW n / as. .. _ L 86:5 or com -- _ 000 .. .09.... r can -- E N.8 maxi com) 8:38 I «8 xan ®ULOL LQQ |.O 0P 5/ JOd (own DISCUSSION In this study we estimated the ATP cost of isometric contractions from time change iii PCr iii spectra. acquired immediately before and after the contractions. This assumes that glycolysis and aerobic metabolism. make no significant contribution to ATP production during the brief time CH? a single tetanus or 5) short burst (Hf twitches. This assumption has been validated in rat hindlimb muscle (Foley gum) Meyer, 1993). The IKE: cost (if contractions measured by gated NMR was the same as that calculated by others from steady—state oxygen consumption measurements. Similarly, PCr cost of contractions measured chemically in mouse muscles was the same as that calculated from recovery oxygen consumption after the contractions (Crow and Kushmerick, 1982). As expected from many previous studies, we found that the ATP cost of both twitch and tetanic contractions under normocapnic conditions was less in slow-twitch than in fast—twitch muscle. These measurements include both the AJT’ cost (if calcium) cycling anmi force (development (i.e. internal shortening), as vmfll. as time cost (Hf isometric force maintenance. However, it appears that the greatest difference between the muscle types is in the cost of force :maintenance. The cost CM? twitch contractions was only about two-fold higher in biceps compared to soleus muscle. In contrast, the ATP cost of a 3 s tetanus in soleus muscle was less timN: 1/3 time ATP cost. red tflimii cells iii bicarbonate- buffered Krebs—Henseleit solution. Perfusate was equilibrated with either 5% C02-95% 02 or 70% C02-30% 02 at 37°C in a SilasticCI tube oxygenator. The muscle was stretched ti) optimal length for maximum isometric twitch force development. Muscles were stimulated try a supramaximal square wave pulses (10—50V, 1 ms) applied from a Grass stimulator. Experimental Series I/Phosphorous NMR The isolated soleus was placed within a 2 cm Helmholtz coil within a custom made 7.4 cm diameter probe. The proximal bone and (distal tendon. were secured. by (custom built DelrimO clamps. The distal clamp was attached to a strain gauge force transducer. Platinwm wire electrodes 134 were securely placed on each end of the muscle for direct stimulation. Muscle temperature was monitored by a thermistor and maintained at 37°C. Phosphorus NMR spectra of isolated. perfused soleus muscles were acquired at 162 DHU1:mount and the distal tendon was attached to a commercial force transducer. Platinum electrodes were placed on each end of the mmscle enmi each mmscle vmms stimulated an: two rates during normocapnia and hypercapnia (0.25, 0.5, 0.75, 1 or 2 Hz). RESULTS Figure 23 shows the PCr changes at stimulation rates of 0.25 and 0.5 Hz of one soleus muscle during normocapnia and hypercapnia. PCr levels decreased with increased stimulation rate under both conditions. PCr levels reached a lower steady—state at the same twitch rate during 1111 normocapnia as compared to hypercapniai The average time constant during normocapnia was 3.4 :t 0.33 minutes and during hypercapnia was 7.66 i 1.0 minutes [Table Ed. The time constant of PCr changes was similar at onset of stimulation and during recovery within each condition. Table 6 summarizes intracellular and extracellular pH and. oxygen consumption an: rest and during stimulation. Resting oxygen consumption was 0.098 i 0.008 umol OZ/min/g wet weight during normocapnia and 0.103 i 0.013 umol OZ/min/g vmi: weight during hypercapnia. Resting intracellular pH was 7.16 i 0.06 during normocapnia and 6.6 i 0.08 during hypercapnia. Intracellular pH was not significantly different from initial pH during the last 5- 10 minutes of stimulation at either pH. However, there was a transient alkalinizaticmi during‘ the first ten Ininutes stimulation at pH 6.6 [Figure 24] that is was not observed at normal pH. There was not a consistent dependence of oxygen consumption on calculated [ADP] concentration during normocapnia and hypercapnia [Figure 25]. There was a linear dependence of steady-state oxygen consumption on steady-state PCr levels [Figure 26] during normocapnia (slope = -0.11, r = 0.97) and hypercapnia (slope = —0.04, r = 0.99). There was a linear relationship between ln[ATP]/[ADP][Pi][H+] at pH 7.2 (slope =—0.78 , r = 0.97, y-intercept : 10.4)and pH (6.6 slope = -0.26, y-intercept = 2.5), r 20.99) [Figure 27]. Figure 23. PCr changes during soleus muscle stimulation (0.25 and 0.5 Hz) and recovery under normocapnic (open circles) and hypercapnic (closed circles) conditions. °—.25 HZ, pH 7.2, ° A—.25 Hz,p1-1 6.6, ‘ ’—.5HZ, pH 7.2 ‘—.5 Hz, pH 6.6 ./. .\./. o/ \OVOZ o/o / o °\o/ 0/0—0 i . /A_A\ /“"°\A/A A / 0 /8 i A/ A L a) ‘/‘\\A/ \‘ o\° ‘/ \ /‘ ‘ ‘ \ /A ‘ \.__. / \A 1 I I I I I 15 20 25 30 35 40 Minutes TABLE 5 PCr time constants (minutes) Normocapnia Hypercapnia onset 3.23i0.5 7.73i0.95* stimulation (n29) (n=7) recovery 3.56i0.44 7.58i2.0* (n=7) (n=7) Values are mean SE, * significantly different from normocapnia (p<0.05). 140 TABLE 6 NORMOCAPNIA HYPERCAPNIA HZ 0 0.25 0.5 1 2 0 0.25 0.5 1 PCr 2.94 3.0 .ATP i0.14 i0.17 pHic 7.16 7.19 7.18 7.17 6.6 6.55 6.57 i0.02 i0.02 i0.03 i0.04 i0.04* i0.03* i0.03* pHec 7.5 6.74 i0.03 i0.06* Q02 0.098 0.295 0.464 0.585 0.848 0.102 0.205 0.26 0.189 umol/ i0.01 i0.04 i0.08 i0.07 i0.2 i0.01 i0.05 i0.01 i0.08 min/g (Values are mean i SE, * significantly different from normocapnia at the same stimulation rate (p<0.05). 141 Figure 24. pH changes during 0.25 and 0.5 Hz stimulation of soleus muscle during normocapnia (N=pH 7.2) and hypercapnia (A=pH 6.6). 0.5 Hz at pH = 7.2 (filled circles); 0.25 Hz at pH = 7.2 (open circles); 0.5 Hz, pH = 6.6 (closed triangles); 0.25 Hz, pH = 6.6 (open triangles). °.25N°.5N 6.25/4 ‘.5A 7.300 _ T T T ’ * - T T I I l ° I . ° I 7'2OO§E§§§£§E§8IEQ§§$§66:6 7.100~- 7000—— 6.900-I 6.800~— 6.700—— T T 0 T I E 1 1 1 E I T T T B'GOoiiiiggléiiiliiiiiii; t tillilliiilii 6500+ 6.400 I I I I P I I I I MlNUTES Figure 25. [ADP] versus oxygen consumption at 0.25 and 0.5 Hz during hypercapnia and 0.25, 0.5 and 1 Hz during normocapnia. Calculated [ADP] from creatine kinase equilibrium assuming apparent Keq = 1.66. At rest [ATP] = 5.03 mM, total Cr = 24.4 mM, and Pi = 10.1 mM from chemical analysis of perfused muscles (Meyer et al., 1985). ph == 6.6 (filled circles); pH = 7.2 (open circles). umol Oz/min/g C)600~— Cl400 ()200 ()000 I44 ° °PH=73 ° °pH=66 l E l i 1/ /l 10 2b 50 4b 5b 60 i0 1 ADP mM X 10‘3 80 Figure 26. Steady—state PCr levels versus oxygen consumption of soleus muscle during rest and stimulation at 0.25 and 0.5 Hz during hypercapnia (pH = 6.6) and 0.25, 0.5, and 1 Hz during normocapnia (pH = 7.2). pH = 6.6 (filled circles, r = 0.99); pH = 7.2 (open circles, r = 0.97) 0600—— .0 4; O O % umol 02/min/g .0 [\D o O 0.000 I46 umol PCr/g “b 10 d)- 11 I47 Figure 27. Steady-state cytoplasmic phosphorylation potential versus oxygen consumption of soleus muscle during rest and stimulation at 0.25 and 0.5 Hz during hypercapnia and 0.25, 0.5 and 1 Hz during normocapnia. Calculated [ADP] from creatine kinase equilibrium assuming apparent Keq = 1.66.. At rest [ATP] = 5.03 mM, total Cr = 24.4 mM, and Pi = 10.1 mM from chemical analysis of perfused muscles (Meyer et al., 1985). ph = 7.2 (open circles, r = 0.97); pH = 6.6 (closed circles, r = 0 99). I48 OTIEQTQQEEVE me e T.— [Oi ,._o__q I /7 I00m0 000.0 I00v0 I000.0 b/qu/ZQ Ioum 149 Discussion Several studies of isolated mitochondria suggest that decreased pH results in decreased maximum oxygen consumption (Westerblad and Lannergren, 1988; Tobin et al., 1972; Chang and Mergner, 1973). Two features of our results appear tx> confirni that acidosis has time same effect on maximum oxygen consumption in intact skeletal muscle. First, l1] our study time time constants for EKH: changes during the onset of stimulation and during recovery were 2- fold longer during acidosis. Because the time constant for PCr changes if; inversely proportional tx> maximum aerobic capacity, this suggests that maximum aerobic capacity was decreased by 2—fold. Second, direct measurements of oxygen consumption showed that it did not significantly increase above 0.189 umol/min/g during acidosis, but reached at least 0.848 umol/min/g under normal pH conditions. Our results are not consistent with a recent study by Nioka, et afl_ (1992), which ch11 not find ea reduction in maximum oxygen consumption during acidosis in intact muscle. They not only found the Vmax of oxidative phosphorylation Ix: be similar‘ between.:normal and ltwv pH stimulation, but also found a dependence of respiration on cytoplasmic [ADP] in intact skeletal muscle. They observed a dependence of work (proportional to oxygen consumption) on [ADP] in a Michelis~Menton fashion with an estimated Km of 26 um, in VlVO. One of their assumptions was that an increase in proton concentration will result in a decrease ISO in PCr levels. Results from our studies (Table 6, see also Chapter 4) (k) not confirm this assumption since at rest, during normocapnia enmi hypercapnia, the EKHQ .ATP, and EH. concentrations are similar. They also use the tension—time integral to estimate oxygen consumption, but used submaximal voltages during stimulation so the fraction of fibers that were actually contracting is obscure, and this may have affected their results. Also, they induced hypercapnia tn/ ventilating tjxe animal \Nlth 20% (X3; for several hours, which most likely introduced several physiological alterations tflmfi: may also lunna complicated the results of the experiment. Our results are not compatible with the simple kinetic ADP theory of respiratory control. At rest, with an experimentally imposed increase in hydrogen ion concentration, you would expect either a lower oxygen consumption rate (if PCr was constant) or a decrease in PCr levels ttf oxygen consumption VMHS constant). [We did not observe 51 significant difference ill PCr levels (n: oxygen consumption rate at rest between normal and low pH conditions. Also, at aum/ given respiration. rate Iduring stimulation, you would expect lower PCr levels and correspondingly a higher [ADP]. EUrthermore, because the maximum oxidative rate was reduced, ADP should have been even higher than predicted from the rfli change alone. In fact, we observed lower cytoplasmic [ADP] at similar oxidative rates during acidic stimulationi These results 15! are difficult to reconcile with control of respiration by total cytoplasmic ADP. Our data is consistent with linear models of the control of respiration with cytoplasmic phosphorylation potential as the signal for oxidative phosphorylation. The time constants were similar during stimulation and recovery at each pH, consistent with predictions of the linear model proposed tn/ Meyer. Steady-state' oxygen. consumption. was linearly related tx3 steady-state PCr levels at kxflji pH values, although the slope during acidosis was reduced over 2-fold. This decrease in slope would be expected since the mitochondrial capacity was reduced by over 50% during hypercapnia. The relationship) between «:ytoplasnur: phosphorylation potential and oxygen consumption was also linear with a significant reduction in y—intercept and slope during acidosis. These results are consistent with the predictions for thermodynamic models, as again, the slope change (XNi be attributed t1) the decreased Initochondrial capacity. The reduction of y—intercept was expected due to the increase :Ui hydrogen itn1 concentration, although the exact attenuation cannot txe determined. because the free energy potential within the mitochondrial may also change with acidosis. The reduction of mitochondrial capacity in intact skeletal muscle tissue with acidosis also has implications for the controversy about the role of acidity in fatigue. 152 Taken together' with time observation. of IX) reduction in maximal force production, our observation that the maximum aerobic capacity is decreased, suggests that the correlation seen between acidosis and fatigue in human studies may be due to the restriction of ATP supply rather than a direct inhibition at the crossbridges. Another interesting feature of our results is the transient alkalinization during the initial stages of stimulation during acidosis. Transient alkalinization at the beginning of contraction has been contributed to the net consumption of protons by PCr hydrolysis (see Equation 3, chapter 1). Because the stoichiometric coefficient increases as pH decreases you would expect a larger transient alkalinization at lower pH. This is exactly what we observed, confirming that the net alkalinization at the beginning of contraction is due to the effect of PCr hydrolysis. In summary our study eliminates total cytoplasmic ADP level as time primary regulator‘ of respiratiCWI but does remain consistent with thermodynamic theories of control of respiration with cytoplasmic phosphorylation as the signal for stimulation of oxidative phosphorylation CHAPTER 6 SUMMARY AND CONCLUSIONS The primary focus of this research was to distinguish between the two prominent models of the control of oxidative phosphorylation. The kinetic model of respiration states that oxidative phosphorylation is limited by ADP availability and should follow a Michaelis— Menton dependence of respiratory rate of cytoplasmic [ADP]. Thermodynamic models of time contndl of respiration argue that the mitochondria is regulated by cytosolic phosphorylation potential. Correlative experiments relating phosphate changes and oxygen consumption coincide satisfactorily vnlji both theories. Yet, kn/ manipulating intracellular pH the theories can be distinguished. In fast-twitch muscle, several studies have confirmed that phosphorylation state, i.e. one of the primary models, controls respiration. Yet, in slow-twitch. muscle some studies suggest control tux substrate' availability. Our initial experiments focused on whether slow-twitch muscle was regulated by phosphorylation state. Our results support that phosphorylation state regulates respiration in slow—twitch muscle in situ although it did not specifically distinguish between the two primary models. 153 154 The remainder of the research focused on distinguishing between the (1“) models cm? the control of respiration by changing the intracellular concentration of hydrogen ion. Previous studies indicated that the relaxation times of isometric contractions were reduced but that maximal force was not attenuated. This suggested that energy cost may be different with twitch contractions during acidosis and therefore it was important to explore this energetic parameter in order to design our experiments to test the two models of the control of respiration during acidosis. We then implemented experiments to determine the energy cost of contraction of fast—and slow-twitch muscle during acidosis. CHM? data showed that energy cost of contraction was reduced during acidosis during twitch contractions but not during tetanus in both muscle types. The reduction of energy cost in skeletal muscle could be attributed to several factors. The velocity of contraction could tme reduced, tflme reactions involved. in contraction inhibited, or calcium cycling attenuated. Although the energy cost could be attributed to force reduction \Nlth tmnlmfli contractions, :maximal force Ioutput was not inhibited. This would suggest that acidosis is not exerting its effects on the crossbridge itself but possibly on calcium handling. Previous studies with ATP depletion have shown a reduction in energy cost without a reduction in force. This energy cost may also be attributable to an affect on calcium handling. This hypothesis could be [55 tested by designing experiments that stimulate the muscle fibers while stretched beyond the length of crossbridge attachment. The muscle would not be able to develop force but enzymes involved in calcium cycling would be activated. iBy measuring energy cxmm: during normal enmi acidic conditions this hypothesis could be addressed. Additional studies could be designed to directly measure the velocity of contraction during acidosis. The final series of experiments were then designed to test the control of oxidative phosphorylation at rates within the maximum aerobic capacity of slow-twitch skeletal muscle. Our data supports the control of respiration by cytosolic phosphorylation potential, a direct prediction of thermodynamic models since respiration is directly dependent on cytosolic phosphorylation state at various pH values. In contrast, our data is not consistent with the simple kinetic theory of control of respiration since there was not 51 consistent dependence (if respiration (Mi [ADP] during acidosis. There was also ea reduction in maximal oxygen consumption with low ph. 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