Dynamics of microvascular oxygen pressure during rest-contraction transition in skeletal muscle of diabetic rats

Type I diabetes reduces dramatically the capacity of skeletal muscle to receive oxygen (QO(2)). In control (C; n = 6) and streptozotocin-induced diabetic (D: n = 6, plasma glucose = 25.3 +/- 3.9 mmol/l and C: 8.3 +/- 0.5 mmol/l) rats, phosphorescence quenching was used to test the hypothesis that, in D rats, the decline in microvascular PO(2) [Pm(O(2)), which reflects the dynamic balance between O(2) utilization (VO(2)) and QO(2)] of the spinotrapezius muscle after the onset of electrical stimulation (1 Hz) would be faster compared with that of C rats. Pm(O(2)) data were fit with a one or two exponential process (contingent on the presence of an undershoot) with independent time delays using least-squares regression analysis. In D rats, Pm(O(2)) at rest was lower (C: 31.2 +/- 3.2 mmHg; D: 24.3 +/- 1.3 mmHg, P < 0.05) and at the onset of contractions decreased after a shorter delay (C: 13.5 +/- 1.8 s; D: 7.6 +/- 2.1 s, P < 0.05) and with a reduced mean response time (C: 31.4 +/- 3.3 s; D: 23.9 +/- 3.1 s, P < 0.05). Pm(O(2)) exhibited a marked undershoot of the end-stimulation response in D muscles (D: 3.3 +/- 1.1 mmHg, P < 0.05), which was absent in C muscles. These results indicate an altered VO(2)-to-QO(2) matching across the rest-exercise transition in muscles of D rats.     

Effect of reactive oxygen species on K+ contractures in the rat diaphragm

Reactive oxygen species (ROS) are postulated toalter low-frequency contractility of the unfatigued and fatigueddiaphragm. It has been proposed that ROS affect contractility throughchanges in membrane excitability and excitation-contraction coupling. If this hypothesis is true, then ROS should alterdepolarization-dependent K⁺contractures. Xanthine oxidase (0.01 U/ml) + hypoxanthine (1 mM) wereused as a source of superoxide anion eliciting oxidative stress ondiaphragm fiber bundles in vitro. Diaphragm fiber bundles from 4-mo-oldFischer 344 rats were extracted and immediately placed in Krebssolution bubbled with 95% O₂-5%CO₂. After 10 min ofequilibration, a K⁺ contracture(Pre; 135 mM KCl) was induced. Fiber bundles were assigned to thefollowing treatment groups: normal Krebs-Ringer (KR; Con) and thexanthine oxidase system (XO) in KR solution. After 15 min of treatmentexposure, a second (Post) K⁺contracture was elicited. Mean time-to-peak tension for contractures was significantly decreased in Post vs. Pre (16.0 ± 0.7 vs. 19.8 ± 1.0 s) with XO; no change was noted with Con. Furthermore, peak contracture tension was significantly higher (31.5%) in the XO groupPost compared with Pre; again, no significant change was found with KR.The relaxation phase was also altered with XO but not with KR.Additional experiments were conducted with application of 1 mMhypoxanthine, with results similar to the Con group. We conclude thatthe application of ROS altered the dynamics ofK⁺ contractures in the ratdiaphragm, indicating changes in voltage-dependent excitation-contraction coupling.

     

The role of nitric oxide in the spatial heterogeneity of basal microvascular blood flow in the rat diaphragm

The effects of N-nitro-L-arginine (L-NOARG) and N^G-monomethyl-L-arginine (L-NMMA) on the spatialdistribution of diaphragmatic microvascular blood flow were assessed in anesthetized, mechanically ventilated rats. Microvascular blood flow was measured after different periods at either a fixed site (Q_stat) or 25 different sites (Q_scan) using computer-aided laser-Doppler flowmetry (LDF) scanning. The value of Q_stat was unaffected after 15–20 min superfusion with any one of the following agents: L-NOARG (0.1 mM), L-NMMA (0.1 mM), L-arg (10 mM). The cumulative frequency histogram of the Q_scan value in the control group displayed a non-Gaussian distribution that was not significantly affected after 15 min superfusion with the vehicle of L-NOARG. Superfusion with either L-NMMA or L-NOARG at 0.1 mM for 15 min displaced the histogram of cumulative frequency to the left, with the median value of blood flow decreasing by 10 to 20%. However, skewness and kurtosis of the distribution of basal Q_scan were unaffected after superfusion of either of the L-arg analogues. Pretreatment with L-arg (10 mM), followed by co-administration of L-arg (10 mM) with L-NOARG (0.1 mM) only partially prevented L-NOARG from exerting this inhibitory effect on the distribution of basal Q_scan, while pretreatment with L-arg in the same manner could prevent L-NMMA from exerting its inhibitory effect. There was a weak but significant linear relationship between the magnitude of basal Q_scan and normalized changes in basal Q_scan after superfusion of either of the L-arg analogues. In conclusion, a basal NO activity is present in the diaphragmatic microvascular bed of rats. LDF scanning rather may yield more vivid information about the extent of overall tissue perfusion than conventional LDF whenever basal NO activity is involved. Moreover, the parallel flow profiles after NO synthase blockade suggest that the spatial inhomogeneity of basal diaphragmatic microvascular blood flow is not dependent on basal NO formation.     

Effects of eccentric exercise on microcirculation and microvascular oxygen pressures in rat spinotrapezius muscle.

A single bout of eccentric exercise results in muscle damage, but it is not known whether this is correlated with microcirculatory dysfunction. We tested the following hypotheses in the spinotrapezius muscle of rats either 1 (DH-1; n = 6) or 3 (DH-3; n = 6) days after a downhill run to exhaustion (90-120 min; -14 degrees grade): 1) in resting muscle, capillary hemodynamics would be impaired, and 2) at the onset of subsequent acute concentric contractions, the decrease of microvascular O(2) pressure (Pmv(o(2))), which reflects the dynamic balance between O(2) delivery and O(2) utilization, would be accelerated compared with control (Con, n = 6) rats. In contrast to Con muscles, intravital microscopy observations revealed the presence of sarcomere disruptions in DH-1 and DH-3 and increased capillary diameter in DH-3 (Con: 5.2 +/- 0.1; DH-1: 5.1 +/- 0.1; DH-3: 5.6 +/- 0.1 mum; both P < 0.05 vs. DH-3). At rest, there was a significant reduction in the percentage of capillaries that sustained continuous red blood cell (RBC) flux in both DH running groups (Con: 90.0 +/- 2.1; DH-1: 66.4 +/- 5.2; DH-3: 72.9 +/- 4.1%, both P < 0.05 vs. Con). Capillary tube hematocrit was elevated in DH-1 but reduced in DH-3 (Con: 22 +/- 2; DH-1: 28 +/- 1; DH-3: 16 +/- 1%; all P < 0.05). Although capillary RBC flux did not differ between groups (P > 0.05), RBC velocity was lower in DH-1 compared with Con (Con: 324 +/- 43; DH-1: 212 +/- 30; DH-3: 266 +/- 45 mum/s; P < 0.05 DH-1 vs. Con). Baseline Pmv(O(2)) before contractions was not different between groups (P > 0.05), but the time constant of the exponential fall to contracting Pmv(O(2)) values was accelerated in the DH running groups (Con: 14.7 +/- 1.4; DH-1: 8.9 +/- 1.4; DH-3: 8.7 +/- 1.4 s, both P < 0.05 vs. Con). These findings are consistent with the presence of substantial microvascular dysfunction after downhill eccentric running, which slows the exercise hyperemic response at the onset of contractions and reduces the Pmv(O(2)) available to drive blood-muscle O(2) delivery.     

Lymphatic vessels in the developing diaphragm of the rat.

Diaphragms of fetal, neonatal and young albino rats have been observed both under light and electron microscopes to examine the presence and distribution of lymphatic vessels and their morphological features. In fetal diaphragms of between 18 and 22 days of gestation, no normal lymphatic vessels can be seen; only after birth, specifically in neonatal and 2-day-old rats, small lymphatic vessels appear; they are in close proximity to the blood vessels in the inner areas of the muscle. As the rats get older, lymphatic vessels are also observed in the subserosa where an abundant connective tissue is present. The fine structure of diaphragmatic lymphatic vessels is different at different ages. In neonatal rats of up to 2 days, the endothelial wall is very thin and often holed. The relationships between contiguous endothelial cells are characterized by simple end-to-end or overlapping structures. The basement membrane is virtually absent. Within the first week of life, the endothelial wall becomes more complex; along the wall, complex interdigitations between two contiguous endothelial cells often touch. A discontinuous basement membrane and collagen and elastic fibers surround the vessels. In the older rats (from 14 to 25 to 140 days), next to the complex interdigitations which characterize the junction between two contiguous endothelial cells, cellular flaps interdigitate forming a channel which opens out either to the exterior or the interior of the vessel. Dense bundles of elastic and collagen fibers are closely apposed to the endothelial wall.     

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.     

The role of the diffusion of oxygen in the respiration of rat diaphragm at different temperatures

Summary 1. Data are presented to support the hypothesis that the respiration rate of hemidiaphragms and tissue slices is restrictedin vitro because oxygen fails to reach the innermost layers of the tissue.2. Calculation of limiting thickness fromWarburg's formula (Equation 1) requires use of a value for QO₂, but the true value is unknown since it is in turn dependent upon thickness. The dilemma is not avoided by making thinner slices because this damages tissues and reduces the QO₂.3. We found that for rat diaphragm a plot of log QO₂ versus 1/T yields a straight line between -6° and 13.5° C, and a line of half that slope from 18° to 38° C.4. Equations are presented for calculating QO₂ at these temperatures, assuming that oxygenation is incomplete above about 18° C. The calculated values agree well with the observed values. Further, QO₂ of diaphragm at 33° C was higher at an oxygen pressure of 2 atmospheres than 1 atmosphere in agreement with theoretical considerations.

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Die Bedeutung der Sauerstoffdiffusion für die Atmung des Rattendiaphragmas bei verschiedenen Temperaturen

Kurzfassung Es werden Ergebnisse vorgelegt, welche die Hypothese untermauern, daß die Respirationsrate von Hemidiaphragmen und Gewebescheiben in vitro eingeschränkt ist, weil der Sauerstoff die innersten Gewebeschichten nicht zu erreichen vermag. Die Errechnung der kritischen Gewebedicke nachWarburgs Formel erfordert die Einsetzung des Wertes für QO₂, der ist aber unbekannt, da er ja selbst wiederum von der Dicke abhängt. Man kann diesem Dilemma nicht dadurch ausweichen, daß man dünnere Scheiben verwendet; denn das würde die Gewebe beschädigen und so den QO₂ herabsetzen. Wir haben festgestellt, daß die Auftragung von log QO₂ gegen 1/T für das Rattendiaphragma eine gerade Linie ergibt zwischen -6° und 13,5° C und eine Linie mit dem halben Anstiegswinkel zwischen 18° und 38° C. Unter der Annahme, daß das Sauerstoffangebot über etwa 18° C unzureichend wird, werden Gleichungen gegeben für die Errechnung des QO₂ bei den oben genannten Temperaturen. Die errechneten Daten stimmen mit den beobachteten Werten gut überein. Bei 33° C war der QO₂-Wert höher bei einem Sauerstoffdruck von 2 Atmosphären als bei einem solchen von 1 Atmosphäre; dieser Sachverhalt steht in Übereinstimmung mit den theoretischen Erörterungen.

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This work was supported in part by a grant from the National Science Foundation.     

Sepsis increases contraction-related generation of reactive oxygen species in the diaphragm.

Recent work indicates that free radicals mediate sepsis-induced diaphragmatic dysfunction. These previous experiments have not, however, established the source of the responsible free radical species. In theory, this phenomenon could be explained if one postulates that sepsis elicits an upregulation of contraction-related free radical formation in muscle. The purpose of the present study was to test this hypothesis by examination of the effect of sepsis on contraction-related free radical generation [i.e. , formation of reactive oxygen species (ROS)] by the diaphragm. Rats were killed 18 h after injection with either saline or endotoxin. In vitro hemidiaphragms were then prepared, and ROS generation during electrically induced contractions (20-Hz trains delivered for 10 min) was assessed by measurement of the conversion of hydroethidine to ethidium. ROS generation was negligible in noncontracting diaphragms from both saline- and endotoxin-treated groups (2.0 +/- 0. 6 and 2.8 +/- 1.0 ng ethidium/mg tissue, respectively), but it was marked in contracting diaphragms from saline-treated animals (19.0 +/- 2.8 ng/mg tissue) and even more pronounced (30.0 +/- 2.8 ng/mg tissue) in diaphragms from septic animals (P < 0.01). This enhanced free radical generation occurred despite the fact that the force-time integral (i.e., the area under the curve of force vs. time) for control diaphragms was higher than that for the septic group. In additional studies, in which we altered the stimulation paradigm in control muscles to achieve a force-time integral similar to that achieved in septic muscles, an even greater difference between control and septic muscle ROS formation was observed. These data indicate that ROS formation during contraction is markedly enhanced in diaphragms from endotoxin-treated septic animals. We speculate that ROS generated in this fashion plays a central role in producing sepsis-related skeletal muscle dysfunction.     

Developmental changes in neuromuscular transmission in the rat diaphragm.

Neuromuscular transmission was studied in diaphragms from rats of three ages, 4-7 days old, 11-12 days old, and adults with the use of an in vitro phrenic nerve-hemidiaphragm preparation. Each hemidiaphragm was stimulated via either muscle or nerve with 1-s stimulus trains at frequencies from 10 to 100 Hz. The patterns of force development obtained in response to the two routes of stimulation were compared for each group. Diaphragms from adults developed maximum force in response to stimulation of approximately 40 Hz with no significant decrease in force at higher frequencies. Within each stimulus train, once peak force was achieved, it was maintained for the remainder of the stimulus and responses to nerve and muscle stimulation were almost identical. In contrast, diaphragms from 4- to 7-day-old rats developed maximum force at approximately 20 Hz; stimulation at greater than or equal to 60 Hz induced significantly less peak force. This decrease in peak force at higher frequencies was significantly larger for nerve than for muscle stimulation. In addition, during each nerve stimulus train diaphragms from 4- to 7-day-old rats were unable to maintain peak force, which decreased at frequencies greater than 20 Hz. The decrease in force reached approximately 50% of peak at stimulation frequencies greater than or equal to 60 Hz. Diaphragms from 11- to 12-day-old rats showed intermediate responses. Based on the responses to phrenic nerve stimulation, we conclude that the neonatal rat diaphragm shows marked neuromuscular transmission failure that is not seen in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic hypoxia modulates diaphragm function in the developing rat.

We studied the effect of chronic hypoxia on contractile properties and neuromuscular transmission in the developing rat diaphragm. We hypothesized that chronic hypoxia delays maturation of neuromuscular transmission. Phrenic nerve hemidiaphragm preparations were harvested from 3- to 26-day-old rats and littermates raised in 9.5% oxygen. Specific force, contraction time, and one-half relaxation time were measured. Each diaphragm was stimulated directly or via its nerve with 1-s trains at 10-100 Hz. Contraction time and one-half relaxation time decreased with advancing age in both groups, with a greater rate of decrease in hypoxic diaphragms. Specific force was lower for hypoxic diaphragms compared with controls. Diaphragms from the 3- to 10-day-old control and hypoxic groups generated less force in response to stimulation at frequencies >40 Hz but did so to a greater degree with nerve stimulation. Nerve stimulation of diaphragms from 11- to 18-day-old hypoxic rats showed a greater decrease in force with increasing frequency compared with age-matched controls. Diaphragms from 19- to 26-day-old rats showed no difference between the hypoxic and control groups. We conclude that chronic hypoxia leads to diaphragms that generate lower specific force as well as to a delayed maturation of mechanisms involved in neuromuscular transmission.     

Dynamic response of the isolated passive rat diaphragm strip.

To further our understanding of the mechanisms underlying chest wall mechanics, we investigated the dynamic response of the isolated passive rat diaphragm strip. Stress adaptation of the tissue was measured from 0.05 to 60 s after subjecting the strips to strain steps of normalized strain amplitudes from 0.005 to 0.04. The tissue resistance (R), elastance (E), and hysteresivity (eta) were measured in the same range of amplitudes by sinusoidally straining the strip at frequencies from 0.03125 to 10 Hz. The stress (T) depended exponentially on the strain (epsilon) and relaxed and recovered linearly with the logarithm of time. E increased linearly with the logarithm of frequency and decreased with increasing amplitude. R fell hyperbolically with frequency and showed an amplitude dependence similar to that of E. To interpret the strong nonlinear behavior, we extended the viscoelastic model of Hildebrandt (J. Appl. Physiol. 28: 365-372, 1970) to include an exponential stress-strain relationship. Accordingly, the step response was described by T - Tr = Tr(e alpha delta epsilon - 1)(1 - gamma log t), where delta epsilon is the strain amplitude, Tr is the initial operating stress, alpha is a measure of the stress-strain nonlinearity, and gamma is the rate of stress adaptation. The oscillatory response of the model was computed by applying Fung's quasi-linear viscoelastic theory. This quasi-linear viscoelastic model fitted the step and oscillatory data fairly well but only if alpha depended negatively on delta epsilon, as might be expected in a plastic material.     

Regional metabolic differences in the rat diaphragm

This study characterized the biochemical properties of the rat diaphragm by measuring the activities of selected citric acid cycle and glycolytic enzymes. The diaphragm was removed from 10 female Sprague-Dawley rats (180 days old) and dissected into five discrete anatomic regions: crural (region 1), left posterior costal (region 2), left anterior costal (region 3), right anterior costal (region 4), and right posterior costal (region 5). Sections were assayed for total protein concentration and the activities of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH). The SDH activity in the crural region was approximately 18% lower (P less than 0.05) than that in any costal region. Furthermore, protein concentration was significantly lower (P less than 0.05) in the crural region compared with all costal regions. In contrast, costal regions 2-5 did not significantly differ from each other in protein concentration or SDH activity. LDH activity did not differ significantly (P greater than 0.05) between regions. Finally, the LDH-to-SDH activity ratio was significantly higher (P less than 0.05) in the crural diaphragm compared with all costal regions. We conclude that the crural region of the rat diaphragm is significantly lower in oxidative capacity than all the costal regions. Investigators who use a rodent model to study diaphragmatic function and plasticity should consider the oxidative heterogeneity of the diaphragm when designing experiments.     

Estimating oxygen transport resistance of the microvascular wall

The problem of diffusion of O(2) across the endothelial surface in precapillary vessels and its utilization in the vascular wall remains unresolved. To establish a relationship between precapillary release of O(2) and vascular wall consumption, we estimated the intravascular flux of O(2) on the basis of published in vivo measurements. To interpret the data, we utilized a diffusion model of the vascular wall and computed possible physiological ranges for O(2) consumption. We found that many flux values were not consistent with the diffusion model. We estimated the mitochondrial-based maximum O(2) consumption of the vascular wall (M(mt)) and a possible contribution to O(2) consumption of nitric oxide production by endothelial cells (M(NO)). Many values of O(2) consumption predicted from the diffusion model exceeded M(mt) + M(NO). In contrast, reported values of O(2) consumption for endothelial and smooth muscle cell suspensions and vascular strips in vitro do not exceed M(mt). We conjecture that most of the reported values of intravascular O(2) flux are overestimated, and the likely source is in the experimental estimates of convective O(2) transport at upstream and downstream points of unbranched vascular segments.     

Extracellular calcium modulates generation of reactive oxygen species by the contracting diaphragm.

Recent studies have indicated that free radicals may play an important role in the development of muscle dysfunction in many pathophysiological conditions. Because the degree of muscle dysfunction observed in some of these conditions appears to be both free radical dependent and modulated by extracellular calcium concentrations, we thought that there may be a link between these two phenomena; i.e., the propensity of a muscle to generate free radicals may be dependent on extracellular calcium concentrations. For this reason, we compared formation of reactive oxygen species (ROS; i.e., free radicals) by electrically stimulated rat diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 trains/s) incubated in organ baths filled with physiological solutions containing low (1 mM), normal (2.5 mM), or high (5 mM) calcium levels. Generation of ROS was assessed by measuring the conversion of hydroethidine to ethidium. We found ROS generation with contraction varied with the extracellular calcium level, with low ROS production (3.18 +/- 0.40 ng ethidium/mg tissue) for low-calcium studies and with much higher ROS generation for normal-calcium (18. 90 +/- 2.70 ng/mg) or high-calcium (19.30 +/- 4.50 ng/mg) studies (P < 0.001). Control, noncontracting diaphragms (in 2.5 mM calcium) had little ROS production (3.40 +/- 0.80 ng/mg; P < 0.001). To further investigate this issue, we added nimodipine (20 microM), an L-type calcium channel blocker, to contracting diaphragms (2.5 mM calcium bath) and found that nimodipine also suppressed ROS formation (2.56 +/- 0.85 ng ethidium/mg tissue). These data indicate that ROS generation by the contracting diaphragm is strongly influenced by extracellular calcium concentrations and may be dependent on calcium transport through L-type calcium channels.     

In vitro functions of the rat diaphragm during postnatal development.

This study was designed to determine the developmental changes in the functional characteristics of the rat diaphragm. A total of 150 animals were studied at 1, 3, 5, 7 and 9 weeks of postnatal age. Body and diaphragm muscle weights were measured. Diaphragm strips were studied in an in vitro preparation to assess muscle contractile and endurance properties. Total diaphragm weight increased considerably, by a factor of 23 over the 9 week-period of study and was highly correlated with body weight (r = 0.93, P less than 0.01). However, the ratio of diaphragm-to-body weight decreased progressively with age. In comparison with those from older animals, diaphragms from 1 and 3 weeks old animals: (1) generated similar force normalized for muscle weight but a lower force normalized for fibre cross-sectional area (P less than 0.05), (2) had longer time-to-peak tension and one-half relaxation times (P less than 0.01) and (3) were more resistant to fatigue (P less than 0.01). The mechanisms underlying the diaphragm functional development were discussed.