Biaxial constitutive relations for the passive canine diaphragm.

Samples of the muscular sheet excised from the midcostal region of dog diaphragms were subjected to biaxial loading. That is, stresses in the direction of the muscle fibers and in the direction perpendicular to the fibers in the plane of the sheet were measured at different combinations of strains in the two directions. Stress-strain relations were obtained by fitting equations to these data. In the direction of the muscle fibers, for strains up to 0.7, stress is a modestly nonlinear function of strain and ranges up to approximately 60 g/cm. In the direction perpendicular to the fibers, the sheet is stiffer and more strongly nonlinear. At a strain in the perpendicular direction of approximately 0.35, stress increases abruptly. The stress-strain relation in the muscle direction is consistent with observations of passive muscle shortening in vivo. However, the stiffness in the perpendicular direction is not high enough to explain the observation that strains in the perpendicular direction in vivo are nearly zero. We conclude that, in the passive diaphragm in vivo, stress in the direction perpendicular to the muscle fibers is small.     

Mechanical advantage of the canine diaphragm

The mechanical advantage (µ) of a respiratorymuscle is defined as the respiratory pressure generated per unit musclemass and per unit active stress. The value of µ can be obtained by measuring the change in the length of the muscle during inflation ofthe passive lung and chest wall. We report values of µ for themuscles of the canine diaphragm that were obtained by measuring thelengths of the muscles during a passive quasistatic vital capacitymaneuver. Radiopaque markers were attached along six muscle bundles ofthe costal and two muscle bundles of the crural left hemidiaphragms offour bred-for-research beagle dogs. The three-dimensional locations ofthe markers were obtained from biplane video-fluoroscopic images takenat four volumes during a passive relaxation maneuver from total lungcapacity to functional residual capacity in the prone and supinepostures. Muscle lengths were determined as a function of lung volume,and from these data, values of µ were obtained. Values of µ arefairly uniform around the ventral midcostal and crural diaphragm butsignificantly lower at the dorsal end of the costal diaphragm. Theaverage values of µ are 0.35 ± 0.18 and 0.27 ± 0.16 cmH₂O · g¹ · kg¹ · cm²in the prone and supine dog, respectively. These values are 1.5-2 times larger than the largest values of µ of the intercostal muscles in the supine dog. From these data we estimate that during spontaneous breathing the diaphragm contributes ~40% of inspiratory pressure inthe prone posture and ~30% in the supine posture. Passiveshortening, and hence µ, in the upper one-third of inspiratorycapacity is less than one-half of that at lower lung volume. The lower µ is attributed primarily to a lower abdominal compliance at highlung volume.

     

Modeling the kinematics of the canine midcostal diaphragm

The hypotheses that the chest wall insertion (CW) is displaced laterally during inspiration and that this displacement is essential in maintaining muscle curvature of the costal diaphragmatic muscle fibers were tested. With the use of data from three dogs, caudal, lateral, and ventral displacements of CW during both quiet, spontaneous inspiration and during inspiratory efforts against an occluded airway were observed and recorded. We have developed a kinematic model of the diaphragm that incorporates these displacements. This model describes the motions of the muscle fibers and central tendon; the displacements of the midplane, muscle-tendon junction (MTJ), CW, and center of the muscle fiber-central tendon arcs are modeled as functions of muscle fiber length. In the model, the center of the fiber arcs and MTJ both move caudally parallel to the midplane during inspiration, whereas CW moves both caudally and laterally. The observed lateral displacement of CW and the observed caudal displacement of MTJ, as functions of muscle fiber length, both approximate well the theoretical displacements that would be necessary to maintain curvature of the fiber arcs. In confirming our hypotheses, we have found that lateral displacement of CW is a mechanism by which changes in the shape of the costal diaphragm, as described by its curvature, are limited.     

Passive mechanics of muscle tendinous junction of canine diaphragm.

The diaphragmatic muscle tendon is a biaxially loaded junction in vivo. Stress-strain relations along and transverse to the fiber directions are important in understanding its mechanical properties. We hypothesized that 1) the central tendon possesses greater passive stiffness than adjacent muscle, 2) the diaphragm muscle is anisotropic, whereas the central tendon near the junction is essentially isotropic, and 3) a gradient in passive stiffness exists as one approaches the muscle-tendinous junction (MTJ). To investigate these hypotheses, we conducted uniaxial and biaxial mechanical loading on samples of the MTJ excised from the midcostal region of dog diaphragm. We measured passive length-tension relationships of the muscle, tendon, and MTJ in the direction along the muscle fibers as well as transverse to the fibers. The MTJ was slack in the unloaded state, resulting in a J-shaped passive tension-strain curve. Generally, muscle strain was greater than that of MTJ, which was greater than tendon strain. In the muscular region, stiffness in the direction transverse to the fibers is much greater than that along the fibers. The central tendon is essentially inextensible in the direction transverse to the fibers as well as along the fibers. Our data demonstrate the existence of more pronounced anisotropy in the muscle than in the tendon near the junction. Furthermore, a gradient in muscle stiffness exists as one approaches the MTJ, consistent with the hypothesis of continuous passive stiffness across the MTJ.     

Mechanics of the canine diaphragm in pleural effusion

Pleural effusion is a complicating feature of many diseases of the lung and pleura, but its effects on the mechanics of the diaphragm have not been assessed. In the present study, radiopaque markers were attached along muscle bundles in the midcostal region of the diaphragm in anesthetized dogs, and the three-dimensional location of the markers during relaxation before and after the stepwise introduction of liquid into the left or right pleural space and during phrenic nerve stimulation in the same conditions was determined using computed tomography. From these data, accurate measurements of diaphragm muscle length and displacement were obtained, and the changes in pleural and abdominal pressure were analyzed as functions of these parameters. The effect of liquid instillation on the axial position of rib 5 was also measured. The data showed that 1) liquid leaked through the dorsal mediastinal sheet behind the pericardium so that effusion was bilateral; 2) effusion caused a caudal displacement of the relaxed diaphragm; 3) this displacement was, compared with passive lung inflation, much larger than the cranial displacement of the ribs; and 4) the capacity of the diaphragm to generate pressure, in particular pleural pressure, decreased markedly as effusion increased, and this decrease was well explained by the decrease in active muscle length. It is concluded that pleural effusion has a major adverse effect on the pressure-generating capacity of the diaphragm and that this is the result of the action of hydrostatic forces on the muscle.     

Ratio of active to passive muscle shortening in the canine diaphragm.

Active and passive shortening of muscle bundles in the canine diaphragm were measured with the objective of testing a consequence of the minimal-work hypothesis: namely, that the ratio of active to passive shortening is the same for all active muscles. Lengths of six muscle bundles in the costal diaphragm and two muscle bundles in the crural diaphragm of each of four bred-for-research beagle dogs were measured by the radiopaque marker technique during the following maneuvers: a passive deflation maneuver from total lung capacity to functional residual capacity, quiet breathing, and forceful inspiratory efforts against an occluded airway at different lung volumes. Shortening per liter increase in lung volume was, on average, 70% greater during quiet breathing than during passive inflation in the prone posture and 40% greater in the supine posture. For the prone posture, the ratio of active to passive shortening was larger in the ventral and midcostal diaphragm than at the dorsal end of the costal diaphragm. For both postures, active shortening during quiet breathing was poorly correlated with passive shortening. However, shortening during forceful inspiratory efforts was highly correlated with passive shortening. The average ratios of active to passive shortening were 1.23 +/- 0.02 and 1.32 +/- 0.03 for the prone and supine postures, respectively. These data, taken together with the data reported in the companion paper (T. A. Wilson, M. Angelillo, A. Legrand, and A. De Troyer, J. Appl. Physiol. 87: 554-560, 1999), support the hypothesis that, during forceful inspiratory efforts, the inspiratory muscles drive the chest wall along the minimal-work trajectory.     

Mechanics of the canine diaphragm in ascites: a CT study.

Ascites causes an increase in the elastance of the abdomen and impairs the lung-expanding action of the diaphragm, but its overall effects on the pressure-generating ability of the muscle remain unclear. In the present study, radiopaque markers were attached to muscle bundles in the midcostal region of the diaphragm in five dogs, and the three-dimensional locations of the markers during relaxation and during phrenic nerve stimulation in the presence of increasing amounts of ascites were determined using a computed tomographic scanner. From these data, accurate measurements of muscle length and quantitative estimates of diaphragm curvature were obtained, and the changes in transdiaphragmatic pressure (Pdi) were analyzed as functions of muscle length and curvature. With increasing ascites, the resting length of the diaphragm increased progressively. In addition, the amount of muscle shortening during phrenic nerve stimulation decreased gradually. When ascites was 100 ml/kg body wt, therefore, the muscle during contraction was longer, leading to a 20-25% increase in Pdi. As ascites increased further to 200 ml/kg, however, muscle length during contraction continued to increase, but Pdi did not. This absence of additional increase in Pdi was well explained by the increase in the diameter of the ring of insertion of the diaphragm to the rib cage and the concomitant increase in the radius of diaphragm curvature. These observations indicate that the pressure-generating ability of the diaphragm is determined not only by muscle length as conventionally thought but also by muscle shape.     

Interaction between the canine diaphragm and intercostal muscles in lung expansion.

Changes in intrathoracic pressure produced by the various inspiratory intercostals are essentially additive, but the interaction between these muscles and the diaphragm remains uncertain. In the present study, this interaction was assessed by measuring the changes in airway opening (DeltaPao) or transpulmonary pressure (DeltaPtp) in vagotomized, phrenicotomized dogs during spontaneous inspiration (isolated intercostal contraction), during isolated rectangular or ramp stimulation of the peripheral ends of the transected C(5) phrenic nerve roots (isolated diaphragm contraction), and during spontaneous inspiration with superimposed phrenic nerve stimulation (combined diaphragm-intercostal contraction). With the endotracheal tube occluded at functional residual capacity, DeltaPao during combined diaphragm-intercostal contraction was nearly equal to the sum of the DeltaPao produced by the two muscle groups contracting individually. However, when the endotracheal tube was kept open, DeltaPtp during combined contraction was 123% of the sum of the individual DeltaPtp (P < 0.001). The increase in lung volume during combined contraction was also 109% of the sum of the individual volume increases (P < 0.02). Abdominal pressure during combined contraction was invariably lower than during isolated diaphragm contraction. It is concluded, therefore, that the canine diaphragm and intercostal muscles act synergistically during lung expansion and that this synergism is primarily due to the fact that the intercostal muscles reduce shortening of the diaphragm. When the lung is maintained at functional residual capacity, however, the synergism is obscured because the greater stiffness of the rib cage during diaphragm contraction enhances the DeltaPao produced by the isolated diaphragm and reduces the DeltaPao produced by the intercostal muscles.     

Role of endothelium-derived relaxing factor in active hyperemia of the canine diaphragm.

To assess the effect of endothelium-derived relaxing factor (EDRF) on diaphragmatic vascular resistance at rest and during contractions, we studied an in situ isolated diaphragm preparation in anesthetized and mechanically ventilated dogs. The arterial supply of the left diaphragm (phrenic artery) was catheterized and perfused with arterial blood at a fixed flow rate. Drugs were infused through a side port of the arterial catheter at 1/100th of the phrenic arterial flow. The inferior phrenic vein was catheterized to complete the isolation from the systemic circulation. Three sets of experiments were performed. In set 1 (n = 3), we infused endothelium-dependent (acetylcholine, ACh) and endothelium-independent (sodium nitroprusside, SNP) dilators at increasing concentrations. ACh and SNP infusion elicited a dose-dependent decline in phrenic vascular resistance (Rphr) at concentrations greater than 10(-8) M and 0.50 micrograms/ml, respectively. In set 2 (n = 15), we infused an inhibitor of EDRF synthesis and release, L-argininosuccinic acid (ArgSA), at increasing concentrations (10(-4), 3 x 10(-4), and 6 x 10(-4) M). ArgSA produced a dose-dependent increase in Rphr. Infusion of another EDRF inhibitor (NG-nitro-L-arginine, LNA, 6 x 10(-4) M) elicited increase in Rphr similar to that induced by ArgSA. In set 3 (n = 25), we infused ArgSA or LNA (6 x 10(-4) M) simultaneously with ACh and SNP and during sustained (2-Hz) contractions of the diaphragm. Both ArgSA and LNA completely reversed ACh vasodilation, whereas SNP vasodilation was reversed by 26 and 11%, respectively. ArgSA or LNA infusion during contractions reversed vasodilation by 48 and 52%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased fatigue of isovelocity vs. isometric contractions of canine diaphragm

A comparison of fatigue as a loss of force with repeated contractions over time was performed in canine respiratory muscle by isometric (nonshortening) and isovelocity (shortening) contractions. In situ diaphragm muscle strips were attached to a linear ergometer and electrically stimulated (30 or 40 Hz) via the left phrenic nerve to produce either isometric (n = 12) or isovelocity (n = 12) contractions (1.5 s) from optimal muscle length (Lo = 8.8 cm). Similar velocities of shortening between isovelocity experiments [0.19 +/- 0.02 (SD) Lo/S] were produced by maximizing the mean power output (Wmax = 210 +/- 27 mW/cm2) that could be developed over 1.5 s when displacement was approximately 0.30 Lo. Initial peak isometric tension was 1.98 kg/cm2, whereas initial peak isovelocity tension was 1.84 kg/mc2 (P less than 0.01) or 93% of initial isometric tension. Fatigue trials of 5 min were conducted on muscles contracting at a constant duty cycle (0.43). At the end of the trials, peak isovelocity tension had fallen to 50% of initial isometric tension (P less than 0.01), whereas peak isometric tension had only fallen by 27%. These results indicate that muscle shortening during force production has a significant influence on diaphragm muscle fatigue. We conclude that the effects of shortening on fatigue must be considered in models of respiratory muscle function, because these muscles typically shorten during breathing.     

Interaction of fatigue and hypercapnia in the canine diaphragm

We studied 10 open-chest dogs and measured the pressure across the diaphragm (Pdi) in each period of the protocol during stimulation at frequencies of 1, 20, 50, and 80 Hz. Three ranges of arterial PCO2 (PaCO2) were examined: less than or equal to 26, 36-50, and greater than or equal to 89 Torr. The diaphragm was fatigued with repetitive phrenic stimulation (30 Hz). During the fatiguing activity, five of the animals were subjected to hypercapnia and the other five to hypocapnia. A frequency-Pdi curve was generated for each period in the protocol. The data show that 1) fatiguing to 50% of the initial Pdi value during hypercapnia was significantly more rapid than during hypocapnia; 2) both the prefatigue and postfatigue mean Pdi values over all interactions of frequency, fatigue, and PaCO2 were unaffected by the fatiguing environment (hypercapnia vs. hypocapnia); 3) the percent reduction of Pdi by hypercapnia was the same at all four frequencies; 4) hypocapnia did not alter either the pre- or postfatigue frequency-Pdi curve; and 5) one-half relaxation time, unaffected by PaCO2, was prolonged by fatigue. We conclude that the hypercapnic diaphragm has less endurance than the hypocapnic diaphragm and that although both fatigue and hypercapnia decrease Pdi, they appear to be separate entities working through different mechanisms.     

Hypoxemia-induced modification of troponin I and T in canine diaphragm.

Impaired muscle function (fatigue) may result, in part, from modification of contractile proteins due to inadequate O(2) delivery. We hypothesized that severe hypoxemia would modify skeletal troponin I (TnI) and T (TnT), two regulatory contractile proteins, in respiratory muscles. Severe isocapnic hypoxemia (arterial partial pressure of O(2) of approximately 25 Torr) in six pentobarbital sodium-anesthetized spontaneously breathing dogs increased respiratory frequency and electromyographic activity of the diaphragm and internal and external obliques, with death occurring after 131-285 min. Western blot analysis revealed proteolysis of TnI and TnT, 17.5- and 28-kDa fragments, respectively, and higher molecular mass covalent complexes, one of which (42 kDa) contained TnI (or some fragment of it) and probably TnT in the costal and crural diaphragms but not the intercostal or abdominal muscles. These modifications of myofibrillar proteins may provide a molecular basis for contractile dysfunction, including respiratory failure, under conditions of limited O(2) delivery.     

Asymmetrical action of the canine diaphragm after single-lung inflation

Single-lung transplantation (SLT) in patients with emphysema leads to a cranial displacement of the diaphragm on the transplanted side and a shift of the mediastinum toward the transplanted lung. The objective of the present study was to assess the effect of unilateral lung inflation on the mechanics of the diaphragm. Two endotracheal tubes were inserted in the two main stem bronchi of six anesthetized dogs, and radiopaque markers were attached along muscle fibers in the midcostal region of the two halves of the diaphragm. The animals were then placed in a computed tomographic scanner, the left or the right lung was passively inflated, and the phrenic nerves were stimulated while the two endobronchial tubes were occluded. As lung volume increased, the fall in airway opening pressure (ΔPao) in the inflated lung during stimulation decreased markedly, whereas ΔPao in the noninflated lung decreased only moderately (P < 0.001). Also, the two hemidiaphragms shortened both during relaxation and during phrenic stimulation, but the ipsilateral hemidiaphragm was consistently shorter than the contralateral hemidiaphragm. In addition, the radius of curvature of the ipsilateral hemidiaphragm during stimulation increased, whereas the radius of the contralateral hemidiaphragm remained unchanged. These observations indicate that 1) in the presence of unilateral lung inflation, the respiratory action of the diaphragm is asymmetric; and 2) this asymmetry is primarily determined by the differential effect of inflation on the length and curvature of the two halves of the muscle. These observations also imply that in patients with emphysema, SLT improves the action of the diaphragm on the transplanted side.     

In vivo length-force relationship of canine diaphragm

Diaphragmatic length was measured by sonomicrometry and transdiaphragmatic pressure (Pdi) by conventional latex balloons in eight dogs anesthetized with pentobarbital sodium under passive conditions and during supramaximal phrenic stimulation. The passive length-pressure relationship indicates that the crural part of the diaphragm is more compliant than the costal part. With supramaximal stimulation the costal diaphragm showed a length-pressure relationship similar in shape to in vitro length-tension curves previously described for the canine diaphragm. The crural part has a smaller pressure-length slope than the costal part in the length range from 80% of optimum muscle length (Lo) to Lo. At supine functional residual capacity (FRC) the resting length (LFRC) of the costal and crural diaphragms are not at Lo. The costal part is distended to 105% of Lo, and crural is shortened to 92% of Lo. Tidal shortening will increase the force output of costal while decreasing that of the crural diaphragm. The major forces setting the passive supine LFRC are the abdominal weight (pressure) and the elastic recoil of the lungs. The equilibrium length (resting length of excised diaphragmatic strips) was 79 +/- 3.6% LFRC for the costal diaphragm and 87 +/- 3.9% LFRC for the crural diaphragm. Similar shortening was obtained in the upright position, indicating passive diaphragmatic stretch at supine LFRC.     

Shape and tension distribution of the active canine diaphragm

Both diaphragm shape and tension contribute to transdiaphragmatic pressure, but of the three variables, tension is most difficult to measure. We measured transdiaphragmatic pressure and the global shape of the in vivo canine diaphragm and used principles of mechanics to compute the tension distribution. Our hypotheses were that 1) tension in the active diaphragm is nonuniform with greater tension in the central tendon than in the muscular regions; 2) maximum tension is essentially oriented in the muscle fiber direction, whereas minimum tension is orthogonal to the fiber direction; and 3) during submaximal activation change in the in vivo global shape is small. Metallic markers, each 2 mm in length, were implanted surgically on the peritoneal surface of the diaphragm at 1.5- to 2.0-cm intervals along the muscle bundles at the midline, ventral, middle, and dorsal regions of the left costal diaphragm and along a muscle bundle of the crural diaphragm. Postsurgery, a biplane videofluoroscopic system was used to determine the in vivo three-dimensional coordinates of the markers at end expiration and end inspiration during quiet breathing as well as at end-inspiratory efforts against an occluded airway at lung volumes of functional residual capacity and at one-third maximum inspiratory capacity increments in volume to total lung capacity. A surface was fit to the marker locations using a two-dimensional spline algorithm. Diaphragm surface was modeled as a pressurized membrane, and tension distribution in the active diaphragm was computed using the ANSYS finite element program. We showed that the peak of the diaphragm dome was closer to the ventral surface than to the dorsal surface and that there was a depression or valley in the crural region. In the supine position, during inspiratory efforts, the caudal displacement of the dorsal region of the diaphragm was greater than that of the dome, and the valley along the crural diaphragm was accentuated. In contrast, at lower lung volumes in the prone posture, the caudal displacement of the dome was greater than that of the crural region. At end of inspiration, transdiaphragmatic pressure was approximately 6.5 cmH2O, and tensions were nonuniform in the diaphragm. Maximum principal stress sigma(1) of central tendon was found to be greater than sigma(1) of the costal region, and that was greater than sigma(1) of the crural region, with values of 14-34, 14-29, and 4-14 g/cm, respectively. The corresponding data of the minimum principal stress sigma(2) were 9-18, 3-9, and 0-1.5 g/cm, respectively. Maximum principal tension was approximately parallel to the muscle fibers, whereas minimum tension was essentially orthogonal to the longitudinal direction of the muscle fibers. In the muscular region, sigma(1) was approximately 3-fold sigma(2), whereas in the central tendon, sigma(1) was only approximately 1.5-fold sigma(2.).     

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.