Adherent primary cultures of mouse intercostal muscle fibers for isolated fiber studies.

Primary culture models of single adult skeletal muscle fibers dissociated from locomotor muscles adhered to glass coverslips are routine and allow monitoring of functional processes in living cultured fibers. To date, such isolated fiber cultures have not been established for respiratory muscles, despite the fact that dysfunction of core respiratory muscles leading to respiratory arrest is the most common cause of death in many muscular diseases. Here we present the first description of an adherent culture system for single adult intercostal muscle fibers from the adult mouse. This system allows for monitoring functional properties of these living muscle fibers in culture with or without electrical field stimulation to drive muscle fiber contraction at physiological or pathological respiratory firing patterns. We also provide initial characterization of these fibers, demonstrating several common techniques in this new model system in the context of the established Flexor Digitorum Brevis muscle primary culture model.

Common rostrocaudal gradient of output from human intercostal motoneurones during voluntary and automatic breathing.

In voluntary breaths, driven through the motor cortex, the pattern of activation in human inspiratory intercostal muscles is unknown. We measured single motor unit behaviour in the first, third, and fifth parasternal intercostal muscles in 5 subjects for 'quiet' and matched 'voluntary' inspirations. In voluntary breaths, the average onset, peak and end discharge rate of 264 inspiratory single motor units was greater in the first interspace compared to caudal spaces (p < 0.05). Relative to the onset of inspiratory flow, the time of recruitment of single motor units and the onset of multiunit activity were also earlier in the first compared to the fifth interspace (p < 0.05). For 215 'common' motor units, peak discharge frequencies were ∼20% higher in voluntary compared to quiet breaths (p < 0.05), due in part, to small differences in the pattern of breathing. A rostrocaudal gradient of motor unit activation across parasternal intercostal muscles was preserved in voluntary and involuntary tasks. A common mechanism may mediate this pattern of recruitment.

Ultra-rapid engineered collagen constructs tested in an in vivo nursery site.

Collagen is a naturally occurring structural protein, highly conserved across species. Conventionally, tissue engineering aims to convert cell-seeded constructs into a tissue-like architecture with biomimetic function. However, cell-mediated remodelling of biomaterial scaffolds in vitro has proved to be slow, costly and difficult to control. We have recently developed a novel process for ultra-rapid engineering of tissue-like constructs without the need for cell-based remodelling. Using plastic compression of type I collagen gels, the densities of collagen and cells together with mechanical properties can be brought controllably to near-tissue levels in minutes rather than weeks/months. We have now implanted these constructs in a test site across intercostal spaces in a rabbit model designed to provide cyclical tensile loading in vivo, to test their integration, cell ingrowth and angiogenic response over 5 weeks. Post-implanted constructs were recovered and tested for host vascularization, inflammatory response and mechanical integrity.

Response of the rabbit diaphragm to tendon vibration.

To evaluate the potential role of diaphragmatic muscle spindles in the act of breathing, we have recorded the electromyograms of the diaphragm and the external intercostal muscle in the third interspace during high-frequency mechanical vibration (50 Hz) of the central tendon in eight anesthetized, spontaneously breathing rabbits. Vibration induced a consistent, clear-cut increase in the inspiratory activity recorded from the external intercostal, thus indicating that the mechanical stimulus applied to the diaphragm was strong enough to trigger muscle spindles at distant sites. However, vibration did not elicit any alteration in costal or crural diaphragmatic activity in any animal. Similarly, when vibration was applied during hyperventilation-induced apnea, activity was recorded in the external intercostal but not in the diaphragm. These observations support the traditional view that the diaphragm is poorly endowed with muscle spindles and that these play little or no significant role in the act of breathing.

Effects of lung volume on parasternal pressure-generating capacity in dogs.

Previous studies have suggested that the optimum length for force generation of the parasternal intercostal (PS) muscles is well above functional residual capacity (FRC). We further explored this issue by examining the pressure-generating capacity of the PS muscles as a function of lung volume in anaesthetized dogs. Upper thoracic spinal cord stimulation (SCS) was used to electrically activate the PS muscles. Changes in airway pressure and parasternal resting length (LR) during airway occlusion were monitored over a wide range of lung volumes during SCS. To assess the effects of parasternal contraction alone, SCS was performed following phrenicotomy and section of the external intercostal, levator costae and triangularis sterni muscles. With increasing lung volume, there were progressive decrements in the capacity of the PS muscles to produce changes in airway pressure. The relationship between PS pressure generation and lung volume was similar to a previous comparable assessment of the external intercostal muscles. The PS muscles shortened during passive inflation and also shortened further (by > 20 % of LR) during SCS. Total shortening (passive plus active) increased progressively with increasing lung volume. Our results indicate that the capacity of the PS muscles to produce changes in airway pressure (a) falls progressively with increasing lung volume and (b) is similar to that of the external intercostal muscles. We speculate that the fall in PS pressure-generating capacity is related, in part, to progressive reductions in end-inspiratory length.

Comparison of Ca(2+) sparks produced independently by two ryanodine receptor isoforms (type 1 or type 3).

The molecular determinants of a Ca(2+) spark, those events that determine the sudden opening and closing of a small number of ryanodine receptor (RyR) channels limiting Ca(2+) release to a few milliseconds, are unknown. As a first step we investigated which of two RyR isoforms present in mammalian embryonic skeletal muscle, RyR type 1(RyR-1) or RyR type 3 (RyR-3) has the ability to generate Ca(2+) sparks. Their separate contributions were investigated in intercostal muscle cells of RyR-1 null and RyR-3 null mouse embryos. A comparison of Ca(2+) spark parameters of RyR-1 null versus RyR-3 null cells measured at rest with fluo-3 showed that neither the peak fluorescence intensity (DeltaF/F(o) = 1.25 +/- 0.7 vs. 1.55 +/- 0.6), spatial width at half-max intensity (FWHM = 2.7 +/- 1.2 vs. 2.6 +/- 0.6 microm), nor the duration at half-max intensity (FTHM = 45 +/- 49 vs. 43 +/- 25 ms) was significantly different. Sensitivity to caffeine (0.1 mM) was remarkably different, with sparks in RyR-1 null myotubes becoming brighter and longer in duration, whereas those in RyR-3 null cells remained unchanged. Controls performed in double RyR-1/RyR-3 null cells obtained by mice breeding showed that sparks were not observed in the absence of both isoforms in >150 cells imaged. In conclusion, 1) RyR-1 and RyR-3 appear to be the only intracellular Ca(2+) channels that participate in Ca(2+) spark activity in embryonic skeletal muscle; 2) except in their responsiveness to caffeine, both isoforms have the ability to produce Ca(2+) sparks with nearly identical properties, so it is rather unlikely that a single RyR isoform, when others are also present, would be responsible for Ca(2+) sparks; and 3) because RyR-1 null cells are excitation-contraction (EC) uncoupled and RyR-3 null cells exhibit a normal phenotype, Ca(2+) sparks result from the inherent activity of small clusters of RyRs regardless of the participation of these RyRs in EC coupling.

Respiratory mechanical advantage of the canine external and internal intercostal muscles.

1. The current conventional view of intercostal muscle actions is based on the theory of Hamberger (1749) and maintains that as a result of the orientation of the muscle fibres, the external intercostals have an inspiratory action on the lung and the internal interosseous intercostals have an expiratory action. This notion, however, remains unproved. 2. In the present studies, the respiratory actions of the canine external and internal intercostal muscles were evaluated by applying the Maxwell reciprocity theorem. Thus the effects of passive inflation on the changes in length of the muscles throughout the rib cage were assessed, and the distributions of muscle mass were determined. The fractional changes in muscle length during inflation were then multiplied by muscle mass and maximum active stress (3.0 kg cm-2) to evaluate the potential effects of the muscles on the lung. 3. The external intercostals in the dorsal third of the rostral interspaces were found to have a large inspiratory effect. However, this effect decreases rapidly both toward the costochondral junctions and toward the base of the rib cage. As a result, it is reversed to an expiratory effect in the most caudal interspaces. The internal intercostals in the caudal interspaces have a large expiratory effect, but this effect decreases ventrally and rostrally, such that it is reversed to an inspiratory effect in the most rostral interspaces. 4. These observations indicate that the canine external and internal intercostal muscles do not have distinct inspiratory and expiratory actions as conventionally thought. Therefore, their effects on the lung during breathing will be determined by the topographic distribution of neural drive.

Mechanical advantage of the human parasternal intercostal and triangularis sterni muscles.

1. Previous studies in dogs have demonstrated that the maximum change in airway pressure (DeltaPao) produced by a particular respiratory muscle is the product of three factors, namely the mass of the muscle, the maximal active muscle tension per unit cross-sectional area ( approximately 3.0 kg cm-2), and the fractional change in muscle length per unit volume increase of the relaxed chest wall (i.e. the muscle's mechanical advantage). In the present studies, we have used this principle to infer the DeltaPao values generated by the parasternal intercostal and triangularis sterni muscles in man. 2. The mass of the muscles and the direction of the muscle fibres relative to the sternum were first assessed in six cadavers. Seven healthy individuals were then placed in a computed tomographic scanner to determine the orientation of the costal cartilages relative to the sternum and their rotation during passive inflation to total lung capacity. The fractional changes in length of the muscles during inflation, their mechanical advantages, and their DeltaPao values were then calculated. 3. Passive inflation induced shortening of the parasternal intercostals in all interspaces and lengthening of the triangularis sterni. The fractional shortening of the parasternal intercostals decreased gradually from 7.7 % in the second interspace to 2.0 % in the fifth, whereas the fractional lengthening of the triangularis sterni increased progressively from 5.9 to 13.8 %. These rostrocaudal gradients were well accounted for by the more caudal orientation of the cartilages of the lower ribs. 4. Since these fractional changes in length corresponded to a maximal inflation, the inspiratory mechanical advantage of the parasternal intercostals was only 2.2-0. 6 % l-1, and the expiratory mechanical advantage of the triangularis sterni was only 1.6-3.8 % l-1. In addition, whatever the interspace, parasternal and triangularis muscle mass was 3-5 and 1-3 g, respectively. As a result, the magnitude of the DeltaPao values generated by a maximal contraction of the parasternal intercostals or triangularis sterni in all interspaces would be only 1-3 cmH2O. 5. These studies therefore confirm that the parasternal intercostals in man have an inspiratory action on the lung whereas the triangularis sterni has an expiratory action. However, these studies also establish the important fact that the pressure-generating ability of both muscles is substantially smaller than in the dog.

Composition of newly forming motor units in prenatal rat intercostal muscle.

We have examined the composition of rat intercostal motor units during the period of late gestation, when most muscle fibres are formed, in order to see the pattern of the contacts initially made between single motoneurons and myotubes. At this early stage, the muscle contains two types of myotubes, primary and secondary myotubes, and a major aim was to see whether individual motoneurons preferentially made contact with a particular myotube type. The technique used to define myotubes contacted by a single motoneuron was anterograde labelling of the neuron, followed by electron microscopic detection of labelled terminals and their postsynaptic targets. We find that prenatal motor units are inhomogeneous with respect to their primary/secondary myotube composition. Most individual motoneurons show many permutations of contact with primary myotubes, secondary myotubes, and undifferentiated cells, including single nerve terminals which contact both primary and secondary myotubes. Our results are interpreted in terms of changes to the composition of both the muscle and of the motor units during the final 5 days of gestation. We demonstrate that motoneurons necessarily make their initial contacts on primary myotubes, but that these are surprisingly sparse. As secondary myotubes appear and become innervated, motor units are at first all similar and all heterogeneous. However, primary myotubes are represented more often in motor units than in the muscle as a whole. This probably reflects the relative densities of polyinnervation of primary vs. secondary myotubes. By embryonic day 20, motor units have become divergent in composition, with some dominated by primary myotubes and others by secondaries. We propose that motoneurons initially establish contacts at random on either myotube type, but then begin to express preference for one type or the other and reorganise their periphery. Refining of motor unit composition towards homogeneity in the postnatal period probably involves other elements, such as mutability of muscle fibre and/or motoneuron characteristics as a function of usage and muscle position, perhaps influenced by sensory feedback mechanisms.

Respiratory effect of the intercostal muscles in the dog.

In a previous paper (J. Appl. Physiol. 73: 2283-2288, 1992), respiratory effect was defined as the change in airway pressure produced by active tension in a muscle with the airway closed, mechanical advantage was defined as the respiratory effect per unit mass per unit active stress, and it was shown that mechanical advantage is proportional to muscle shortening during the relaxation maneuver. Here, we report values of mechanical advantage and maximum respiratory effect of the intercostal muscles of the dog. Orientations of the intercostal muscles in the third and sixth interspaces were measured. Mechanical advantages of the muscles in these interspaces were computed by computing their shortening from these data and data in the literature on rib displacement. We found that parasternal internal intercostals and dorsal external intercostals of the upper interspace have large inspiratory mechanical advantages and that dorsal internal intercostals of the lower interspace and triangularis sterni have large expiratory mechanical advantages. Mass distributions in the two interspaces were also measured, and maximum respiratory effects of the muscles were calculated from their mass, mechanical advantage, and the value for maximum stress in skeletal muscle. Estimated maximum respiratory effects of the inspiratory and expiratory muscle groups of the entire rib cage were tested by measuring the maximum inspiratory pressures that were generated by the parasternal and external intercostals acting alone. Measured pressures, -13 cmH2O for the parasternals and -11 cmH2O for the external intercostals, agreed well with the computed values.

Rostrocaudal variation of fiber type composition in rat intercostal muscles.

We used the histochemical stain for ATPase to compare the fiber-type composition of rat internal and external intercostal muscles from thoracic (T) segments 2-5, 8, and 11. At each level, type II fibers were more numerous than type I fibers, type II B fibers were more numerous than II A fibers, and type I fibers were more numerous in external than in internal intercostals. However, fiber type composition varied from segment to segment. For example, the proportion of type II A fibers increased in a rostrocaudal gradient in internal but not external intercostals, and type I fibers were more prevalent at rostral and caudal than at intermediate levels in both internal and external intercostals. These results provide a basis for interpreting previous physiological and molecular studies which have compared intercostal muscles from different segmental levels.

Cholinesterase activity of motor end plate in human skeletal muscle.

The activity and properties of cholinesterase of the motor end plate in human intercostal muscle were studied in the isolated muscle membrane. This preparation was used because cholinesterase activity of the membrane preparation was localized in the motor end plate without contamination of cholinesterase of other muscle components. Under the experimental conditions, cholinesterase in a human end plate hydrolyzed 1.21 x 10(8) molecules of acetylcholine per msec, which is smaller than hydrolysis of 2.69 x 10(8) by a motor end plate of rat intercostal muscle. Studies with cholinesterase inhibitors and specific substrates indicated that about 90% of cholinesterase of human motor endplates is acetylcholinesterase, and about 10% is pseudocholinesterase. The end plate cholinesterase had an optimal pH of 7.8 and a Michaelis-Menten constant of 4.15 mmoles/liter, and was stable at 4 degrees C for at least 4 wk. Motor end plates were estimated to contain only about 2% of the total cholinesterase activity of human intercostal muscle, compared with about 20% in rat tibialis anterior muscle. The difference is due to the lower cholinesterase activity of the motor end plate and higher cholinesterase activity of non-end plate components in human muscle than in rat muscle. The isolated muscle membrane provides a useful preparation for the study of the properties of motor end plate in human skeletal muscle.