The cellular effects of carbon monoxide in the airway.

The notion of inhaled carbon monoxide (CO) being a toxic chemical under all circumstances is currently being challenged, as recent research has suggested that low concentrations of CO may have therapeutic value, especially in the airway. This review evaluates CO's effects on cellular functions that may result in beneficial outcomes in the settings of airway disease, inflammation, and injury. CO can modulate the stress response system of the cell by decreasing levels of reactive intermediates over time, produced by mitochondrial iNOS and NADPH oxidase. Intracellular stress-induced response factors (e.g., HIF-1 and HSP- 70) are induced in response to CO, possibly facilitating more rapid and effective defenses, in response to subsequent stressors. CO also can trigger changes in cellular functions downstream, protecting the cells from stress-associated events promoted in the airway, as a result of disease or injury, including reducing rates of apoptosis, proliferation, and inflammatory cellular infiltration, as well as preventing an imbalance in the extracellular matrix composition. CO has also been associated with maintaining homeostasis of ions essential for normal cellular functions (e.g., Na+, Fe2+,3+). CO also targets cell-specific functions of the airway, such as reduction of contractility of airway smooth muscle cells, and preservation of the innate defense mechanism of airway epithelial cells. Further understanding of CO's effects on fundamental cellular functions in the airway will likely hold significant value in future considerations of CO's role in airway therapy and health.

Part 3. Assessment of genotoxicity and oxidative stress after exposure to diesel exhaust from U.S. 2007-compliant diesel engines: report on 1- and 3-month exposures in the ACES bioassay.

Human health hazards due to diesel exhaust (DE*) exposure have been associated with both solvent and combustion components. In the past, diesel engine exhaust components have been linked to increased mutagenicity in cultures of Salmonella typhimurium and mammalian cells (Tokiwa and Ohnishi 1986). In addition, DE has been shown to increase both the incidence of tumors and the induction of 8-hydroxy-deoxyguanosine adducts (8-OHdG) in ICR mice (Ichinose et al. 1997). Furthermore, DE is composed of a complex mixture of polycyclic aromatic hydrocarbons (PAHs) and particulates. One such PAH, 3-nitrobenzanthrone (3-NBA), has been identified in DE and found in urban air. 3-NBA has been observed to induce micronucleus formation in DNA of human hepatoma cells (Lamy et al. 2004). The purpose of the current research, which is part of the Advanced Collaborative Emissions Study (ACES), a multidisciplinary program being carried out by the Health Effects Institute and the Coordinating Research Council, is to determine whether improvements in the engineering of heavy-duty diesel engines reduce the oxidative stress and genotoxic risk associated with exposure to DE components. To this end, the genotoxicity and oxidative stress of DE from an improved diesel engine was evaluated in bioassays of tissues from Wistar Han rats and C57BL/6 mice exposed to DE. Genotoxicity was measured as strand breaks using an alkaline-modified comet assay. To correlate possible DNA damage found by the comet assay, measurement of DNA-adduct formation was evaluated by a competitive enzyme-linked immunosorbent assay (ELISA) to determine the levels of free 8-OHdG found in the serum of the animals exposed to DE. 8-OHdG is a specific modified base indicating an oxidative type of DNA damage to DNA nucleotides. In addition, a thiobarbituric acid reactive substances (TBARS) assay was used to assess oxidative stress and damage in the form of lipid peroxidation in the hippocampus region of the brains of DE-exposed animals. Results from the comet assay showed no significant differences in rats between the control and exposed groups (P = 0.53, low exposure; P = 0.92, medium exposure; P = 0.77, high exposure) after 1 month of DE exposure. There were no differences between sexes in the responses of rats to these exposures. Likewise, there were no significant differences found after 3 months of exposure. Similarly, no significant differences were found between the mice exposed for 1 and 3 months to DE, nor were any differences found between sexes. Measurements of 8-OHdG in both mice and rats showed no significant difference among DE exposure groups (P = 0.46, mice; P = 0.86, rats). In mice, measured 8-OHdG was lower in the 3-month group than the 1-month group. In rats, the inverse was true. In mice, no significant differences in the levels of lipid peroxidation, as measured by TBARS, were found between the controls and DE exposure groups (P = 0.92), nor were there any differences between sexes. In rats, comparisons between the control and low-exposure groups approached significance, but no significant differences were found between the other DE exposure groups. Additionally, in rats, there were no significant differences between the 1- and 3-month DE exposure groups.

Predicting worsening asthma control following the common cold.

The asthmatic response to the common cold is highly variable, and early characteristics that predict worsening of asthma control following a cold have not been identified. In this prospective multicentric cohort study of 413 adult subjects with asthma, the mini-Asthma Control Questionnaire (mini-ACQ) was used to quantify changes in asthma control and the Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21) to measure cold severity. Univariate and multivariable models were used to examine demographic, physiological, serological and cold-related characteristics for their relationship to changes in asthma control following a cold. Clinically significant worsening of asthma control was observed following a cold (mean+/-SD increase in mini-ACQ score of 0.69+/-0.93). Univariate analysis demonstrated that season, centre location, cold duration and cold severity measurements were all associated with a change in asthma control. Multivariable analysis of the covariates available within the first 2 days of cold onset revealed that the day 2 and cumulative sum of day 1 and 2 WURSS-21 scores were significant predictors of the subsequent changes in asthma control. In asthmatic subjects, cold severity within the first 2 days can be used to predict subsequent changes in asthma control. This information may help clinicians prevent deterioration in asthma control following a cold.

Increased nitric oxide production by airway cells of sensitized and challenged IL-10 knockout mice.

The anti-inflammatory cytokine interleukin (IL)-10 suppresses inducible nitric oxide synthase (iNOS); therefore, NO production should increase in the absence of IL-10. Production of NO (as nitrite) by bronchoalveolar lavage cells of IL-10 knockout ((-/-)) mice was assessed after ovalbumin sensitization and airway challenge (S/C) and was compared with the IL-10-sufficient, wild-type (WT) C57Bl6. Eosinophil recruitment occurred in S/C WT and IL-10(-/-) mice, suggesting allergic airway inflammation. Alveolar macrophages (per g mouse) were unchanged (approximately 3x10(4) cells) with the exception of a doubling in the S/C IL-10(-/-) mice (approximately 6x10(4) cells, P<0.05). NO production (per million cells) was doubled in cells from S/C IL-10(-/-) (15.3 microM) mice compared with WT (7.6 microM, P<0.05). Inhibition of iNOS by L-N(5)-(1-iminoethyl)-ornithine reduced NO production in all S/C mice, confirming that the increase was a result of up-regulation of iNOS. We conclude that IL-10 is a critical cytokine regulating iNOS in murine airway cells and that its absence can lead to up-regulation of iNOS and development of allergic airway inflammation.

Power fatigue of the rat diaphragm muscle.

We hypothesized that decrements in maximum power output (W(max)) of the rat diaphragm (Dia) muscle with repetitive activation are due to a disproportionate reduction in force (force fatigue) compared with a slowing of shortening velocity (velocity fatigue). Segments of midcostal Dia muscle were mounted in vitro (26 degrees C) and stimulated directly at 75 Hz in 400-ms-duration trains repeated each second (duty cycle = 0.4) for 120 s. A novel technique was used to monitor instantaneous reductions in maximum specific force (P(o)) and W(max) during fatigue. During each stimulus train, activation was isometric for the initial 360 ms during which P(o) was measured; the muscle was then allowed to shorten at a constant velocity (30% V(max)) for the final 40 ms, and W(max) was determined. Compared with initial values, after 120 s of repetitive activation, P(o) and W(max) decreased by 75 and 73%, respectively. Maximum shortening velocity was measured in two ways: by extrapolation of the force-velocity relationship (V(max)) and using the slack test [maximum unloaded shortening velocity (V(o))]. After 120 s of repetitive activation, V(max) slowed by 44%, whereas V(o) slowed by 22%. Thus the decrease in W(max) with repetitive activation was dominated by force fatigue, with velocity fatigue playing a secondary role. On the basis of a greater slowing of V(max) vs. V(o), we also conclude that force and power fatigue cannot be attributed simply to the total inactivation of the most fatigable fiber types.

Enhancement of adult muscle regeneration by primary myoblast transplantation.

Extensor digitorum longus muscles (EDL) of SCID mice were induced to undergo degeneration-regeneration subsequent to orthotopic, whole-muscle transplantation. Two days after transplantation some of these muscles received injections of primary myoblasts derived from EDL muscles of transgenic mice, which express nuclear localizing beta-galactosidase under the control of the myosin light-chain 3F promoter and enhancer. Nine weeks after transplantation, regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received no further treatment and to unoperated EDL muscles in order to determine the effect of myoblast transfer on muscle regeneration. Many myofibers containing donor derived myonuclei could be identified in the regenerated muscles that had received exogenous myoblasts. The mass of the muscles subjected to transplantation only was significantly less (31% less) than that of unoperated muscles. The addition of exogenous myoblasts to the regenerating EDL resulted in a muscle mass similar to that of unoperated muscles. The absolute twitch and tetanic tensions and specific twitch and tetanic tensions of transplant-only muscles were 28%, 36%, 32%, and 41%, respectively, of those of unoperated muscles. Myoblast transfer increased the absolute twitch and tetanic tensions of the regenerated muscles by 65% and 74%, respectively, and their specific twitch and tetanic tensions were increased by 41% and 48%, respectively. These data suggest a possible role for the addition of exogenous, primary myoblasts in the treatment of traumatized and/or diseased muscles that are characterized by myofiber loss.

Growth hormone improves body mass recovery with refeeding after chronic undernutrition-induced muscle atrophy in aging male rats.

The effects of growth hormone (GH) administration and refeeding after chronic undernutrition (UN) were investigated in Fischer 344 male rats aging into senescence (24.5 mo of age) during UN initiated at 12.5 mo of age that produced muscle atrophy and a 50% decrease in body mass. Muscle mass, protein, myosin heavy-chain (MHC) composition and circulating testosterone levels were measured and compared to controls with free access to food. Within 9 wk, refeeding + GH restored body mass to control levels, whereas it was still decreased with refeeding alone. By 24.5 mo of age, refeeding alone restored body mass, while addition of GH resulted in overshoot. UN uniformly decreased mass of the gastrocnemius, extensor digitorum longus, soleus and diaphragm muscles to 50-60% of controls. Refeeding and refeeding + GH restored these losses with some overshoot of gastrocnemius muscle suggesting hypertrophy. UN more than doubled slow Type I MHC composition and approximately halved fast Type IIB and IIX MHC in the deep gastrocnemius muscle while it increased Type IIA MHC in the diaphragm. Refeeding and refeeding + GH reversed these shifts. MHC shifts in the extensor digitorum longus and soleus muscles were not statistically significant, whereas UN increased fast Type IIA MHC followed by decrease with refeeding + GH. UN decreased testosterone levels to nearly zero followed by restoration with refeeding and refeeding + GH. We conclude that the phenotype of mixed-MHC muscles such as the gastrocnemius and diaphragm are most affected by chronic UN, which is reversible with refeeding and refeeding + GH. These alterations were associated with changes in circulating testosterone, which may be a key regulatory element in these processes.

Growth hormone restores aged diaphragm myosin composition and performance after chronic undernutrition.

The effects of growth hormone (GH) on diaphragm muscle myosin heavy chain (MHC) composition and mechanical performance were investigated in Fischer 344 male rats aged to senescence (24.5 mo of age). Chronic undernutrition (UN), refeeding (RF), and RF+GH were compared with ad libitum feeding by using a model of UN that produced a 50% decrease in body weight over a 12-mo period. The effect of aging was assessed by comparing MHC composition of ad libitum-fed rats at 12 and 24.5 mo of age. At senescence, significant decreases in slow type I (-23%) and fast type IIA (-31%) MHC had occurred with aging. Conversely, UN over this aging period increased types I (32-73%) and IIA (22-23%) MHC and decreased fast types IIB (32-54%) and IIX (30-31%) MHC. RF and RF+GH reversed these shifts back toward control values. At senescence, maximal specific force, maximal velocity, and specific power capacity were not different across treatment groups. During repetitive isotonic contraction trials, the diaphragms of UN rats maintained power production over time (54% of initial power at 60 s), whereas the power production of diaphragms of ad libitum-fed rats fell to 0% (P < 0.05). In comparison with UN rats, the diaphragms of RF and RF+GH rats produced 23 (not significant) and 11% (P < 0.05) of initial power, respectively, suggesting that RF+GH treatment restored performance characteristics after UN. We conclude that RF+GH can reverse alterations in MHC composition and mechanical performance produced by chronic UN in the aged rat diaphragm.

Influence of nitric oxide on vascular resistance and muscle mechanics during tetanic contractions in situ.

Studies of the effect of nitric oxide (NO) synthesis inhibition were performed in the isometrically contracting blood-perfused canine gastrocnemius-plantaris muscle group. Muscle blood flow (Q) was controlled with a pump during continuous NO blockade produced with either 1 mM L-argininosuccinic acid (L-ArgSA) or N(G)-nitro-L-arginine methyl ester (L-NAME) during repetitive tetanic contractions (50-Hz trains, 200-ms duration, 1/s). Pump Q was set to match maximal spontaneous Q (1.3-1.4 ml. min(-1). g(-1)) measured in prior, brief (3-5 min) control contraction trials in each muscle. Active tension and oxygen uptake were 500-600 g/g and 200-230 microl. min(-1). g(-1), respectively, under these conditions. Within 3 min of L-ArgSA infusion, vascular resistance across the muscle (R(v)) increased significantly (from approximately 100 to 300 peripheral resistance units; P < 0.05), whereas R(v) increased to a lesser extent with L-NAME (from approximately 100 to 175 peripheral resistance units; P < 0.05). The increase in R(v) with L-ArgSA was unchanged by simultaneous infusion of 0.5-10 mM L-arginine but was reduced with 1-3 microg/ml sodium nitroprusside (41-54%). The increase in R(v) with L-NAME was reversed with 1 mM of L-arginine. Increased fatigue occurred with infusion of L-ArgSA; active tension and intramuscular pressure decreased by 62 and 66%, whereas passive tension and baseline intramuscular pressure increased by 80 and 30%, respectively. These data indicate a possible role for NO in the control of R(v) and contractility within the canine gastrocnemius-plantaris muscle during repetitive tetanic contractions.

Refeeding reverses cardiac myosin shifts induced by undernutrition in aged rats: modulation by growth hormone.

Growth hormone (GH) was administered (1 mg/day, i.p., 7.5 months) to male Fischer 344 rats, in conjunction with refeeding (RF) after chronic undernutrition (UN), from middle age (17 months old) to senescence (24.5 months old), during which cardiac myosin heavy chain (MHC) profiles were determined by gel-electrophoresis. At 17 months of age, respective MHC-alpha and -beta composition was 74 and 26% in the right ventricle (RV), and 58 and 42% in the left ventricle (LV), of ad libitum-fed controls. At 24.5 months of age, MHC profiles of controls were shifted toward the MHC-beta isoform in both RV (alpha=53%, beta=47%) and LV (alpha=40%, beta=60%), indicating a significant effect of aging on MHC composition in both ventricles. At 17 months of age, 7.5 months of UN likewise resulted in a shift toward the MHC-beta isoform in both RV (alpha=31%, beta=69%) and LV (alpha=22%, beta=78%) as compared to controls, indicating a significant effect of UN in both ventricles. Continued UN into senescence maintained these altered profiles in both ventricles, at 24.5 months of age (RV: alpha=35%, beta=65%; LV: alpha=24%, beta=76%). RF+GH administered from middle age into senescence restored the MHC composition in both ventricles (RV: alpha=57%, beta=43%; LV: alpha=43%, beta=57%), to that of the controls. RF, alone, likewise reversed ventricular MHC composition toward that of MHC-alpha, but appeared to overcompensate (RV: alpha=67%, beta=33%; LV: alpha=46%, beta=54%), surpassing the control and RF+GH profiles, significantly in the RV. These data suggest that GH is a modulator of restoration of cardiac MHC composition, when RF is administered to counter the effects of chronic UN, in the aging rat heart.

Diaphragm myosin heavy chain composition shifts in aging chronically-undernourished Fischer 344 male rats.

The effects of chronic undernutrition (UN) on respiratory muscle were investigated during UN producing a 50% decrease in body weight over a prolonged period (45 weeks) in Fischer 344 male rats. This model focused on progressive, aging-related changes in myosin heavy chain (MHC) profile over time, in which the confounding effects of early development and late senescence were avoided. With aging toward late adulthood (68 weeks), MHC composition of control diaphragms was shifted, with decreased type I (slow) and IIA MHC, and increased type IIB and IIX (fast) MHC. UN produced a divergence of this profile, with an increase in type I and IIA MHC, and decreased type IIX MHC. UN diaphragms in vitro were more resistant to loss of active force with fatigue, during repetitive contractions. However, passive tension rose disproportionately during fatigue, suggesting increased fatigability. We conclude that the observed changes in diaphragm mechanical function are consistent with the UN-induced shifts in MHC composition; however, the elevated passive tension with fatigue suggests additional UN-induced changes in mechanical properties that are possibly detrimental to respiratory muscle function. The UN-dependent divergence in phenotype and mechanical properties may be amplified by aging-related shifts in muscle MHC composition over time, in the control group.

Mechanical and metabolic determination of VO2 and fatigue during repetitive isometric contractions in situ.

Repetitive isometric tetanic contractions (1/s) of the canine gastrocnemius-plantaris muscle were studied either at optimal length (Lo) or short length (Ls; approximately 0.9 . Lo), to determine the effects of initial length on mechanical and metabolic performance in situ. Respective averages of mechanical and metabolic variables were (Lo vs. Ls, all P < 0.05) passive tension (preload) = 55 vs. 6 g/g, maximal active tetanic tension (Po) = 544 vs. 174 (0.38 . Po) g/g, maximal blood flow (Q) = 2.0 vs. 1.4 ml . min-1 . g-1, and maximal oxygen uptake (VO2) = 12 vs. 9 micromol . min-1 . g-1. Tension at Lo decreased to 0.64 . Po over 20 min of repetitive contractions, demonstrating fatigue; there were no significant changes in tension at Ls. In separate muscles contracting at Lo, Q was set to that measured at Ls (1.1 ml . min-1 . g-1), resulting in decreased VO2 (7 micromol . min-1 . g-1), and rapid fatigue, to 0.44 . Po. These data demonstrate that 1) muscles at Lo have higher Q and VO2 values than those at Ls; 2) fatigue occurs at Lo with high VO2, adjusting metabolic demand (tension output) to match supply; and 3) the lack of fatigue at Ls with lower tension, Q, and VO2 suggests adequate matching of metabolic demand, set low by short muscle length, with supply optimized by low preload. These differences in tension and VO2 between Lo and Ls groups indicate that muscles contracting isometrically at initial lengths shorter than Lo are working under submaximal conditions.

Mass and functional capacity of regenerating muscle is enhanced by myoblast transfer.

Morphology and functional capacity of homotopically transplanted extensor digitorum longus muscles (EDL) of adult SCID mice that received 1 x 10(6) myoblasts [stably transfected to express nuclear localizing beta-galactosidase under the control of the myosin light-chain 3F promoter/enhancer] 2 days posttransplantation were evaluated 9 weeks after transplantation, to determine whether the injection of exogenous myoblasts had an effect on muscle regeneration. Regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received (a) no further treatment, or (b) sham injection of the vehicle (without myoblasts) and to unoperated EDL. Nine weeks after myoblast transfer, myofibers containing donor-derived nuclei could be identified after staining with X-gal solution. Judging from its size and poor functional performance compared to muscles subjected to transplantation only, sham injection provided a secondary trauma to the regenerating muscle from which it failed to fully recover. In comparison to the sham-injected muscle, the myoblast-injected muscles weighed 61% more and had 50% more myofibers and 82% more cross-sectional area occupied by myofibers at the muscles' widest girths. Their absolute twitch and tetanic tensions were threefold and twofold greater, respectively, and their specific twitch and tetanic tensions were 71% and 50% greater, respectively, than those of sham-injected muscles. In many parameters, the regenerating muscle subjected to myoblast transfer equaled or exceeded those of muscles that were transplanted only (received only one trauma). Absolute twitch and tetanic tensions were 73% and 65% greater, respectively, and specific twitch tensions of the muscles receiving myoblasts were 50% greater than forces generated by muscles subjected to whole-muscle transplantation only.

Combined myofibrillar and mitochondrial creatine kinase deficiency impairs mouse diaphragm isotonic function.

Creatine kinase (CK) is an enzyme central to cellular high-energy phosphate metabolism in muscle. To characterize the physiological role of CK in respiratory muscle during dynamic contractions, we compared the force-velocity relationships, power, and work output characteristics of the diaphragm (Dia) from mice with combined myofibrillar and sarcomeric mitochondrial CK deficiency (CK[-/-]) with CK-sufficient controls (Ctl). Maximum velocity of shortening was significantly lower in CK[-/-] Dia (14.1 +/- 0.9 Lo/s, where Lo is optimal fiber length) compared with Ctl Dia (17.5 +/- 1.1 Lo/s) (P < 0.01). Maximum power was obtained at 0.4-0.5 tetanic force in both groups; absolute maximum power (2,293 +/- 138 W/m2) and work (201 +/- 9 J/m2) were lower in CK[-/-] Dia compared with Ctl Dia (2,744 +/- 146 W/m2 and 284 +/- 26 J/m2, respectively) (P < 0.05). The ability of CK[-/-] Dia to sustain shortening during repetitive isotonic activation (75 Hz, 330-ms duration repeated each second at 0.4 tetanic force load) was markedly impaired, with CK[-/-] Dia power and work declining to zero by 37 +/- 4 s, compared with 61 +/- 5 s in Ctl Dia. We conclude that combined myofibrillar and sarcomeric mitochondrial CK deficiency profoundly impairs Dia power and work output, underscoring the functional importance of CK during dynamic contractions in skeletal muscle.

Regional intramuscular pressure development and fatigue in the canine gastrocnemius muscle in situ.

Intramuscular pressure (PIM) was measured simultaneously in zones of the medial head of the gastrocnemius-plantaris muscle group (zone I, popliteal origin; zone II, central; zone III, near calcaneus tendon) to determine regional muscle mechanics during isometric tetanic contractions. Peak PIM averages were 586, 1,676, and 993 mmHg deep in zones I, II, and III and 170, 371, and 351 mmHg superficially in zones I, II, and III, respectively. During fatigue, loss of PIM across zones was greatest in zone III (-81%) and least in zone I (-60%) when whole muscle tension loss was -49%. Recovery of PIM was greatest in zone III and least in zone II, achieving 86% and 67% of initial PIM, respectively, when tension recovered to 89%. These data demonstrate that 1) regional mechanical performance can be measured as PIM within a whole muscle, 2) PIM is nonuniform within the canine gastrocnemius-plantaris muscle, being greatest in the deep central zone, and 3) fatigue and recovery of PIM are dissimilar across regions. These differences suggest distinct local effects that integrate to determine whole muscle mechanical capacity during and after intense exercise.

Oxytocin-mediated recruitment of slowly cycling cross bridges and isometric force in rat myometrium.

The relationship between cross-bridge cycling rate and isometric stress was investigated in rat myometrium. Stress production by myometrial strips was measured under resting, K+ depolarization, and oxytocin-stimulated conditions. Cross-bridge cycling rates were determined from measurements of maximal unloaded shortening velocity, using the quick-release method. Force redevelopment after the quick release was used as an index of cross-bridge attachment. With maximal K+ stimulation, stress increased with increased cross-bridge cycling (+76%; P < 0.05) and attached cross bridges (+112%; P < 0.05). Addition of oxytocin during K+ stimulation further increased stress (+30%; P < 0.05). With this force component, the cross-bridge cycling rate decreased (-60%; P < 0.05) similar to that under resting conditions. Attached cross-bridges did not increase with this additional stress. The results suggest two distinct mechanisms mediating myometrial contractions. One requires elevated intracellular calcium and rapidly cycling cross bridges. The other mechanism may be independent of calcium and appears to be mediated by slowly cycling cross bridges, supporting greater unit stress.

Blood flow and pressure relationships which determine VO2max.

The role of O2 delivery in regulating VO2max has been studied in an isolated gastrocnemius-plantaris muscle preparation contracting in situ; recent data addressing this issue are presented. VO2 increases nonlinearly with stimulation frequency reaching a peak at 5 twitches.s-1 or 1 tet.s-1 (200 ms trains, 50 imp.s-1). Further increases in stimulation frequency result in a lower VO2. Measured VO2 maxima are less than predicted VO2 capacity, and peak VO2 during tetanic contractions is greater than that during twitches. Above 150 imp.min-1, VO2 is directly related to the level of blood flow attained as VO2/Q (arterial-venous O2 difference) is fixed by some unknown mechanism. Increasing blood flow, with a pump, during 1.s-1 tetanic contractions increases O2 diffusive conductance and peak VO2. When O2 delivery is reduced, ischemic hypoxia appears to result in more rapid reductions in muscle performance than hypoxic hypoxia because of decreases in perfusion pressure and Q. 31P-NMR studies reveal that reductions in creatine phosphate and energy charge are similar between ischemia and hypoxia suggesting a common regulator, O2. We conclude that VO2max is limited by O2 delivery as a result of a limited and uneven distribution of muscle blood flow. These limitations appear secondary to mechanical restraints imposed by contraction duty cycle and vascular compression.

Muscle blood flow and distribution determine maximal VO2 of contracting muscle.

During repetitive contractions, the VO2 of the dog gastrocnemius-plantaris muscle rose with the contraction frequency up to a maximal value and then decreased as contraction frequency was increased further. PVO2 was constant over most of the contraction frequency range. Reducing perfusion pressure/blood flow reduced VO2max with a constant PVO2. During these maneuvers the diffusion conductance, DCO2 (VO2/PVO2), changed with VO2. Raising the perfusion pressure/flow with a pump increased VO2 with a small rise in PVO2 so that DCO2 also increased. Removing tension from the muscle between contractions elevated VO2 and DCO2 without a change in perfusion pressure. Hypoxemia decreased VO2 with a decrease in PVO2; DCO2 remained constant. A three-compartment mathematical model, based on microsphere measurements of regional flow, was used to illustrate how regional flow variations may exist, and how they are poorly revealed in the mixed whole-muscle venous blood. The model shows VO2.g-1 strongly related to flow. As VO2.g-1 increased as Q.g-1 increased, extraction decreased, and DCO2 increased.

Preload release increases blood flow and decreases fatigue during repetitive isotonic muscle contractions.

The effects of preload on blood flow (Q), O2 uptake (VO2), and fatigue were investigated in the canine gastrocnemius-plantaris muscle in situ. Repetitive (1 contraction/s, 200 ms duration) afterloaded (0.25-0.3 maximal active isometric tension) isotonic tetanic contractions were performed in high-preload (HP; 69 g/g, n = 5), low-preload (LP; 35 g/g, n = 6), and preload-release (PR; 0 g/g, n = 5) experiments. Maximal Q values (1.0, 1.6, and 2.1 ml.min-1.g-1, P < 0.05 for all comparisons) and Q2 delivery (8, 13, and 17 mumol.min-1.g-1, P < 0.05 for all comparisons) increased significantly with decreasing preload. The maximal VO2 of HP was 7.2 mumol.min-1.g-1, which is significantly lower than both LP (10.5 mumol.min-1.g-1, P < 0.05) and PR values (11.4 mumol.min-1.g-1, P < 0.05); these differences were sustained through 20 min of contractions. Fatigue, measured as a loss of power production, was 63, 37, and 23% at 20 min of contractions in HP, LP, and PR, respectively, indicating significantly less fatigue with decreasing preload (P < 0.05 for all comparisons). These data demonstrate that the preload, present as the level of passive tension maintained between contractions, can influence Q, VO2, and fatigue during repetitive isotonic tetanic contractions of muscle in situ by a mechanically determined metabolic modulation of dynamic muscle performance.

Metabolic and work capacity of skeletal muscle of PFK-deficient dogs studied in situ.

Mechanical and metabolic relationships of muscle lacking phosphofructokinase (PFKD) activity were compared with muscle having normal phosphofructokinase (NORM) activity by using the gastrocnemius-plantaris muscle group with isolated circulation in situ. Muscle contractile properties were similar in both groups. Initial power output (W) during repetitive tetanic (200 ms, 50 impulses/s) isotonic contractions was similar in both groups; however, W declined significantly more (30-80%) in PFKD than in NORM muscle over time, with a constant O2 uptake (VO2)/W. Despite similar O2 and substrate delivery, PFKD muscle had a lower VO2 (42-55%), less glucose uptake, similar free fatty acid uptake, and lactic acid uptake rather than output, during contractions. Muscle venous H+ concentration, strong ion difference, and PCO2 increased during contractions, the magnitude of change being smaller in PFKD muscle. Elevating arterial lactate concentration before contractions in PFKD muscle resulted in significant improvements in W and VO2 without altering the acid-base exchange at the muscle. Increasing O2 delivery by increasing arterial O2 concentration in PFKD dogs did not improve W or VO2. We conclude that, despite no inherent mechanical or contractile differences, PFKD muscle has a severely limited oxidative capacity and exaggerated fatigue and blood flow responses to contractions due to limited substrate metabolism resulting from the inability to utilize glycogen and/or glucose.