Evolution and Functional Differentiation of the Diaphragm Muscle of Mammals

Abstract

Symmorphosis is a concept of economy of biological design, whereby structural properties are matched to functional demands. According to symmorphosis, biological structures are never over designed to exceed functional demands. Based on this concept, the evolution of the diaphragm muscle (DIAm) in mammals is a tale of two structures, a membrane that separates and partitions the primitive coelomic cavity into separate abdominal and thoracic cavities and a muscle that serves as a pump to generate intra-abdominal (P_ab ) and intrathoracic (P_th ) pressures. The DIAm partition evolved in reptiles from folds of the pleural and peritoneal membranes that was driven by the biological advantage of separating organs in the larger coelomic cavity into separate thoracic and abdominal cavities, especially with the evolution of aspiration breathing. The DIAm pump evolved from the advantage afforded by more effective generation of both a negative P_th for ventilation of the lungs and a positive P_ab for venous return of blood to the heart and expulsive behaviors such as airway clearance, defecation, micturition, and child birth. © 2019 American Physiological Society. Compr Physiol 9:715-766, 2019.

Figure 1:

Cells require energy substrates to function, even at basal levels of low metabolic work (A). The major energy substrate for ATP production is oxygen (O₂), supplies by gaseous exchange within the lung. Other major energy substrates include carbohydrates and fats, delivered via the arterial circulation from sources within the gastrointestinal tract and liver. At the level of the capillaries, cells uptake these energy substrates and excrete waste products of ATP generation, including carbon dioxide (CO₂), which is returned to the atmosphere during gaseous exchange. Metabolic work increases the requirement for ATP, and during muscle activity, gaseous exchange must be increased in order to cope with the increased demand for O₂ and removal of CO₂. B shows the allometric relationship of basal metabolic rate against body weight. Adapted from data within refs , , , , , , , and .

Figure 2:

The septum transversum is found in all vertebrates, and serves as a partition to separate the heart and pericardium from the peritoneum of the abdominal cavity. It forms in relation to the pericardium and folds caudally in association with the liver. In fish, the septum transversum divides the pericardial and peritoneal cavities. In amphibians and reptiles, it divides the pericardial cavity and the common pleuroperitoneal cavity, which contains the lungs, urogenital and visceral abdominal organs. In the mammals, the septum transversum, now muscularized in the form of the diaphragm muscle, extends the entire span of the body cavity, forming a separation between the collection of the pericardial and pleural (containing the lung) cavities and the peritoneal cavity (i.e. a thoracic cavity and an abdominal cavity). By partitioning separate abdominal and thoracic cavities, smaller relative abdominal and thoracic spaces are created, which improves the efficiency of P_ab and P_th generation. Note that in fish, amphibians and most reptiles, this partition is incomplete and less efficient. In advanced reptiles and all mammals, a complete separation of the thoracic and abdominal cavities is achieved, with this separation being muscularized in the case of mammals, the diaphragm muscle. After Kingsley [351].

Figure 3:

Embryonic timeline of body cavity formation in humans. By week 5, pleuroperitoneal membranes are a pair of membranes which gradually separate the pleural and peritoneal cavities, produced as the pleural cavities expand by invading the body wall. The pleuropericardial membranes separate the developing heart from the developing lung buds. Pleuropericardial membranes initially appear as small folds or ridges projecting into the primitive undivided thoracic cavity. The folds contain the common cardinal veins which drain the primitive venous system into the sinus venosus of the primitive heart. During week 6, the edges of these membranous folds have fused with the dorsal mesentery of the esophagus and with the septum transversum to separate the pleural and pericardial cavities. As the heart descends and the pleural cavities expand, the membranes are drawn out in a mesentery like fold that extends from the lateral wall. By week 7, these membranes fuse with the mesoderm ventral to the esophagus, forming a single pericardial cavity and left and right pleural cavities. During week 8, the lung buds grow into the medial walls of the nascent pleural cavities; and the pleural cavities expand around the heart into the body wall. After Pansky [496].

Figure 4:

The evolution of air breathing occurred before the evolution of aspiration breathing. The simplified clade diagram shows that ray-finned fishes (actinoptarygii) employed buccal pump ventilation of the gills. Air breathing lungfish (dipnoi) and amphibians also employed this mechanism of ventilation, which involved buccal expansion to draw air into the oral cavity under negative pressure, followed by buccal compression, with a closed mouth and nares to inflate lungs under positive pressure. Turtles (testudines), scaled reptiles (lepidosauria), crocodiles (archosauria – also includes birds) and mammals all breathe using an aspiration mechanism, whereby the lungs are inflated with a negative intra-thoracic pressure. Importantly, aspiration breathing requires ribs being attached to the vertrebrae. The ribs serve to stabilize the coelomic cavity for walking and act as a bellows for aspiration. The evolution of aspiration breathing occurred some point between emergence of amphibians and reptiles, though in some reptiles, buccal breathing mechanisms are employed intermittently.

Figure 5:

Anatomical schematic showing placement of the solid-state pressure catheters for measurement of esophageal intra-thoracic (P_th) and gastric intra-abdominal (P_ab) pressures in rodents. As the DIAm contracts it moves caudally, creating a negative P_th and inspiratory airflow and a positive P_ab. The resulting transdiaphragmatic pressure (P_di = P_ab – P_th) reflects DIAm force generation.

Figure 6:

The muscles of the thoracic wall provide for radial expansion of the chest wall (the external intercostal muscles), cranial expansion of the ribcage (parasternals, sternocleidomastoid and scalene) and caudal expansion of the thoracic cavity (the diaphragm muscle). In concert these provide for the generation of negative intra-thoracic pressures. The muscles of the abdominal walls include the lateral (internal intercostal, internal oblique, external oblique and transverse abdominal muscles) and ventral walls (the rectus abdominis and transverse thoracis), that serve to increase intra-abdominal pressured by compressing the abdomen. The cranial wall of the abdominal cavity is provided by the diaphragm muscle, which when activated reduces the cranial extent of the abdominal cavity, thus increasing intra-abdominal pressures.

Figure 7:

Single multinucleated (green, propidium iodide myonuclear stain) diaphragm muscle fibers (A) exhibit striated sarcomeric structures (membrane stained with RH414, red). These striations comprise of the thin (actin) and thick filaments (myosin) seen in transmission electron micrographs (B). In response to Ca²⁺, overlapping filaments undego cross-bridge formation, driving the production of force. The mechanical cycling of cross bridges causes a force vector from the Z-disc towards the midline of the sarcomere (C). The electron micrograph of a skeletal muscle cross-section (D) shows the classical myofilament lattice spacing. The larger structures are the myosin filaments, each surrounded by six smaller actin filaments (E). Each actin filament is surrounded by three myosin filaments, and two actin filaments are shared by any given myosin filament. Thus arrangement allows for the doubled hexagonal crystalline array of the myofilament lattice, the distance between the center of the myosin filament and the center of an adjacent actin filament is 12 nm. The distance between the centers of adjacent myosin filaments is 40 nm. Adapted from elements within ref and .

Figure 8:

Excitation-contraction coupling is mediated by the sarcoplasmic reticulum, which acts as a store for Ca²⁺ (A). Within the T-tubules, depolarization waves activate dihydropyridine receptors (DHPRs; voltage sensitive L-type Ca2+ channels), which in turn induces an initial ryanodine receptor (RyR) mediated Ca²⁺ release. A positive feedback process induces further Ca²⁺-induced Ca²⁺ release. This process rapidly floods the cytosolic space surrounding contractile proteins with free Ca²⁺, eventually binding to troponin C, removing the steric hindrance and allowing for cross-bridge formation (B). This regulation of myosin attachment to actin also involves troponin T (TnT), which binds the troponin complex to the tropomyosin molecule and troponin I (TnI) that actually blocks the actin binding site. Release of Ca²⁺ to the cytosol is followed by the development of muscle fiber force (C). The Ca²⁺ binding of troponin complexes regulate the attachment of myosin heads to actin and thus regulate force generation, as reflected by a sigmoidal force-Ca²⁺ relationship. Adapted from elements within refs , and .

Figure 9:

In the DIAm, maximal power output is achieved at ~30% of maximal shortening velocity and ~30% of maximal force generation/load (A). The force a muscle fiber generates is related to its length (B). At sub-optimal lengths, not all actin and myosin elements are able to form cross-links, thus force is limited. At optimal length (L_o), maximal actin and myosin cross bridge formation is possible, thus force generation is maximal. Beyond this length, passive tension (stretch) causes reduction in the possible number of cross-bridges able to be formed and active force generation is reduced. Adapted from elements within refs , and .

Figure 10:

Peak ATP consumption rates (2.7 nmol mm⁻³ s⁻¹) occur at peak power output for diaphragm muscle (top graph). Within different diaphragm muscle fiber types, ATP consumption varies according to MyHC concentration (increasing with increased MyHC) and the apparent rate of cross-bridge detachment (lower graph). Adapted from ref .

Figure 11:

Cross-bridges cycle between a strongly bound and an unbound state during force generation and contraction. Cross-bridge cycling determines rates of cross-bridge attachment (f_app) and detachment (g_app). The rate of force development (f_app) and cross-bridge cycling (g_app) in MyHC_2A-expressing fibers is greater than that of MyHC_SLOW- expressing fibers. Adapted from ref .

Figure 12:

During the determination phase of myogenesis, stem cells divide and are committed to myoblasts, a process requiring expression of the regulatory factors MyoD and Myf-5. Subsequently, myoblast that are non-proliferative fuse to form primary myotubes, which eventually become muscle fibers that express MyHC_SLOW. Primary myotubes that have further fusion of non-proliferative myoblasts become secondary myotubes. These myotubes develop into muscle fibers capable of expressing MyHC_SLOW or any of the fast MyHC isoforms.

Figure 13:

The prenatal determination of phrenic nerve emergence, axon terminal vesicular release and neuromuscular junction specialization is independent of muscle cells until embryonic (E) day 13, where it contacts the primordial DIAm (A). Muscle development and synaptic formations then enter into a state of co-dependence with nerve (B), with the establishment of adult muscle fiber types, innervation ratios and the lack of polyneuronal innervation (synapse elimination) occurring postnatally (B). Adapted from ref .

Figure 14:

Pre- and postnatal developmental changes in maximum DIAm specific force (normalized to muscle cross-sectional area) show a ~20-fold increase from embryonic to adult (A). The maximum velocity of DIAm shortening increases ~5-fold in the same timespan (B). Adapted from refs and .

Figure 15:

The abdominal view of the DIAm (A) clearly shows the vena cava and aortic vessels passing through the central tendon and aortic hiatus, respectively. The esophagus passes through the left and right crus of the DIAm, forming the esophageal sphincter. The crus inset on the lumbar vertebrae caudal to the thoracic vertebrae where the ribs articulate, helping to create the domed structure of the DIAm. After Downey [152]. There is a marked difference in phrenic nerve branching and neuromuscular junction (NMJ) innervation of the diaphragm in small and large species, such as rats and humans (B). In rats, the NMJ innervation is very linear. In humans and other larger species, there is less linearity to the NMJ innervations and more numerous and elaborate secondary phrenic nerve branching.

Figure 16:

The DIAm consists of four major anatomical divisions, the sternal, costal and crural muscular regions and the central tendon. The DIAm is innervated via the phrenic nerve, emanating from C₃-C₆. Innervation of the DIAm exhibits somatotopy, where phrenic motor neurons from the cranial regions of the phrenic motor pool innervate the more ventral sternal and ventral costal regions. Phrenic motor neurons from the more caudal portion of the phrenic motor neuron pool innervate the dorsal costal and dorsal crural regions of the DIAm. This figure has been amended from ref .

Figure 17:

Signaling between the phrenic nerve and the DIAm occurs across the neuromuscular junction. Neurofilament (red), labels phrenic nerve axons (presynaptic), with α-bungarotoxin labeling the postsynaptic acetylcholine receptors (green) on muscle fibers (with MyHC_2B expressing fibers labeled in blue) (A). The ultrastucture of the neuromuscular junction is illustrated using electron microscopy, with synaptic vesicles (acetylcholine) released at active zones via exocytosis into the junctional fold (B). Adapted from ref .

Figure 18:

Phrenic motor neurons exist bilaterally in the cervical spinal cord (~C₃-₆) and are readily labeled by intrapleural injection of fluorescently-conjugated cholera-toxin subunit B (A). Phrenic motor neurons have extensive dendritic arborisations (B) and exhibit a large heterogeneity in somatic surface areas (C), passive electrical properties which largely determine motor unit recruitment order.

Figure 19:

Different motor unit types exhibit different size and complexity of presynaptic axon terminal (red) and postsynaptic acetylcholine receptor (green) structures. Note that neuromuscular junctions of type IIx and/or IIb muscle fibers are markedly larger and more complex compared to neuromuscular junctions of type I or IIa muscle fibers.

Figure 20:

At E16, many postsynaptic acetylcholine receptor clusters are not innervated yet by axons (arrows in A). At birth, polyneuronal innervation of one acetylcholine receptor cluster by two motor axons is clearly shown (arrow in B).

Figure 21:

Different DIAm motor unit types are distinguished by their intrinsic, mechanical and fatigue properties and are classified as type S, FR, FInt and FF (A). Within a particular motor unit, all muscle fibers are homogeneous, as evidenced by myosin heavy chain (MyHC) expression (B). In the DIAm of most species, type I and IIa diaphragm muscle fibers have smaller cross-sectional areas than those of type IIx and IIb fibers (C). Differences in specific force between different fiber types is related to the different MyHC content per half sarcomere and differing unitary forces produced by different MyHC isoforms (D). Adapted from elements within refs and .

Figure 22:

The initial model for force-frequency coding and DIAm motor unit recruitment was based on direct measurements performed in cats (A). Onset activation frequency for type S and FR motor units is ~8 Hz and maximal activation range is ~25 Hz (blue portion). For type FInt and FF motor units, onset is ~12 Hz and maximal activation ~60 Hz (green portion). The steepest portion of the force-frequency curve occurs between 10 and 30 Hz for all types of motor units in the diaphragm muscle (B). Individual motor unit recordings show that motor units with larger discharges are recruited after those with smaller discharges (C). Motor units are recruited in an orderly fashion with type S > FR > FInt > FF (D). Recruitment of type S and FR motor units is sufficient to accomplish ventilatory behaviors, including eupnea, response to hypoxia/hypercapnea and breathing against an occluded airway. To accomplish higher force expulsive/straining behaviors, such as coughing, sneezing, vomiting and defecation, recruitment of higher-force generating type FInt and FF motor units is necessitated. In general, diaphragm motor units operate in the steep portion of the frequency-coding curve (i.e., between 50–100% activation). Adapted from elements within refs .

Figure 23:

The order of motor unit recruitment is related to the intrinsic properties of motor neurons. Of these the most important is motor neuron surface area, the size principle. Neuronal membrane acts as a capacitor, with total membrane capacitance (C) primarily determined by membrane surface area (A) and distance between the membrane lipid bilayer (d). As membrane bilayers are unchanged, a larger neuronal surface area (A₂ > A₁) will increase capacitance. For a given synaptic input (I_syn), the excitability of the membrane (dV_m/dt) is inversely related to neuronal capacitance. Thus, smaller motor neurons (type S and FR) with low capacitance are more excitable and recruited before larger motor neurons (type FInt and FF).

Figure 24:

Neuromotor control of DIAm ventilatory and expulsive/straining behaviors requires cortical, brainstem and spinal cord centers. Ventilatory behaviors are the most-well characterized of these systems and require the recruitment of predominantly type S and FR motor units. Cortical pathways are able to modulate the eupnic rhythm by interactions with the ventilatory central pattern generator (CPG) or directly via synapses onto phrenic motor neurons (PhMNs). The ventilatory CPG activates brainstem premotor neurons that in turn innervate the PhMNs. Activity of PhMNs during ventilation is also modulated (directly and indirectly) by spinal cord ascending tracts and interneurons. Brainstem chemoreceptors and lung mechanoreceptors regulate the activity of premotor neurons, and act to increase premotor neuron discharge (and thus PhMN activity) during hypoxia/hypercapnia. In the case of expulsive/straining behaviors, the majority of control centers are located within the spinal cord, and recruitment of type FInt and FF motor units (higher-force producing units) is necessitated. Some cortical control of the PhMNs and spinal expulsive/straining CPG may be evident, but rectal and vaginal stretch receptors also elicit strong P_ab generation. There may be shared spinal premotor neurons within the spinal cord for PhMNs and abdominal muscle MNs, and a variety of ascending projections may facilitate the coordinated activity of all MNs involved in expulsive/straining maneuvers. Overall, expulsive behaviors result in near maximal co-contractions of the DIAm and abdominal wall muscles.