Respiratory dysfunction due progressive weakness of the respiratory muscles, particularly the diaphragm, is a major cause of death in the neuromuscular disease (NMD) X-linked myotubular myopathy (XLMTM). Methods of respiratory assessment in patients are often difficult, especially in those who are mechanically ventilated. The naturally occuring XLMTM dog model exhibits a phenotype similar to that in patients and can be used to determine quantitative descriptions of dysfunction as clinical endpoints for treatment and the development of new therapies. In experiments using respiratory impedance plethysmography (RIP), XLMTM dogs challenged with the respiratory stimulant doxapram displayed significant changes indicative of diaphragmatic weakness.
This article serves to describe the need for animal models and, more specifically, a canine model, to accurately define and establish clinical end points for the treatment of neuromuscular disease (NMD) in humans. Characterization of respiratory involvement in NMDs is needed, and the canine model can be used to asses, define, characterize and establish efficacy of new therapeutic modalities.
Respiratory assessment in NMD patients
Although the genetic etiology, pathophysiology, and disease course varies vastly between the inherited NMDs, a common clinical feature is impaired ventilatory function. Respiratory muscle dysfunction increases the rate of pulmonary complications and death, and is a major factor in respiratory failure. Respiratory failure is the primary cause of mortality for many inherited NMDs.
Although a common attribute of neuromuscular compromise is the predominance of respiratory insufficiency, the pathology, clinical signs, onset, and response to medical management vary vastly between NMD diagnoses. For example, patients with Pompe disease may be distinguished from other NMDs by a relatively earlier involvement of ventilatory weakness, with a preservation of locomotor function. As a result, the time course and pattern of ventilatory muscle weakness differs between the NMDs. Vital capacity declines by greater than 8% per year in Duchenne muscular dystrophy (DMD) after the age of 12, yet inspiratory strength remains preserved to a greater degree than expiratory strength. In DMD and amyotrophic lateral sclerosis (ALS), maximal inspiratory and expiratory pressures correlate equally with unassisted cough peak flows. By contrast, the rate of decline in global ventilatory function varies vastly between patients with Pompe disease, but is characterized by predominant diaphragm muscle weakness. Intercostal and diaphragm muscle involvement are differentially affected among spinal muscular atrophy variants and may not occur at all in the mildest, adult-onset phenotype.
Pulmonary pressures generally decline prior to a loss of forced expiratory volumes in most NMDs, making strength a more sensitive index of respiratory function. Because both the inspiratory and expiratory muscles can be affected by NMD, it is important to conduct regular assessments of ventilatory muscle function. Different aspects of ventilatory muscle function can be discerned during assessments of resting tidal breathing, respiratory challenges with external loads, hypercapnia or hypoxia, muscle strength and endurance testing, and forced expiratory maneuvers, as well as during evoked contractions.
Inspiratory muscle weakness
Early declines in respiratory muscle strength may go undetected by patients with NMD, because their overall activity levels often decline concurrently, and tidal breathing requires only a small proportion of the pressure capacity of the system. As a result, significant decreases in strength may occur before an appreciable change in vital capacity. In patients with generalized NMD, a 25% or greater decrease in vital capacity between the upright and supine postures has been shown to predict significant diaphragm muscle weakness. The weakened inspiratory pump has been associated with a stiffened chest wall and increased lung elastic loads, microatelectasis, reduced long volumes with restrictive pulmonary function impairment, CO 2 retention, and rapid, shallow breathing pattern. In addition, patients with NMD and respiratory muscle weakness may have a greater susceptibility to fatigue.
Weakness of the respiratory pump can be measured as a decrease in its pressure-generating capacity. In many NMDs the strength of the respiratory pump is poorly correlated with that of the limb muscles, and should be evaluated separately. Volitional pressure generation can be expressed as the maximal transdiaphragmatic pressure, described later.
Maximal Inspiratory Pressure
Maximal inspiratory pressure (MIP) measures the global pressure capacity of the inspiratory muscle pump at the mouth or tracheostomy opening. It is a noninvasive test with established reference values for age and gender, in both children and adults. MIP can be influenced by lung volume and is typically measured from residual volume. Accurate measurements may also be challenging for patients who are mechanically ventilated or have difficulty consistently following commands. In these cases, valid maximal efforts can be obtained with short bouts of inspiratory occlusions.
Nasal Sniff Pressure
Alternatively, nasal sniff pressures mimic a natural motor function, and maximal sniff maneuvers correlate well with MIP measurements at both the mouth and esophagus. In patients with NMD and severe ventilatory weakness, sniff pressure correlates well with restrictions in vital capacity related to pulmonary restrictive disease and/or progression of diaphragm dysfunction. Whereas MIP is a static maneuver, sniff is quasi-isometric, and different inspiratory muscle recruitment patterns have been noted during each maneuver. Thus, the combined use of MIP and maximal sniff pressures may prove valuable for accurate diagnosis of weakness and longitudinal follow-up of patients with NMD.
Transdiaphragmatic Pressure
The presence of specific diaphragm muscle weakness can be determined by the transdiaphragmatic pressure (Pdi) gradient. Balloon catheters or microtransducers placed in the esophagus (Pes) and stomach (Pga) can be used to estimate abdominal and pleural pressures. In healthy individuals, Pes becomes more negative during inspiration whereas Pga becomes more positive. Pdi can be evaluated during maximal voluntary inspiratory maneuvers or during contractions evoked by electrical or magnetic stimulation. In addition, Pdi during tidal breathing and inspiratory challenges may identify the fatigability of the diaphragm muscle. The use of esophageal manometry can provide a greater insight into specific diaphragm muscle weakness but the procedure is invasive, and catheter placement may be poorly tolerated by patients.
Diaphragmatic paralysis results in an upward movement of the diaphragm, resulting in a negative Pga during inspiration. As a result, the Pdi approaches zero. This process is accompanied by asynchronous, paradoxic breathing. The timing and extent of the chest and abdominal wall movements can be quantified during tidal breathing, as well as maximal efforts, by respiratory inductance plethysmography.
Evoked Phrenic Motor Unit Function
Nonvolitional assessments of diaphragm contractile function reduce the variability of voluntary, maximal-effort maneuvers and remove the role of patient cooperation from the examination. Diaphragm contractions can be elicited by supramaximal electrical or magnetic stimulation of the phrenic nerves and be measured by Pdi or electromyogram (EMG) responses. In patients with DMD and severe diaphragm dysfunction, diaphragm electrophysiological responses remained present even when Pdi responses to phrenic stimulation could not be detected. The diaphragm motor responses revealed a normal to elevated ventilatory drive despite an undetectable Pdi. In conjunction with needle EMG activity at rest and during graded contractions, the electrophysiological features elicited during phrenic stimulation can differentiate patients with an isolated neural pathology from those with a purely muscular impairment or a mixed pathology.
Inspiratory muscle weakness
Early declines in respiratory muscle strength may go undetected by patients with NMD, because their overall activity levels often decline concurrently, and tidal breathing requires only a small proportion of the pressure capacity of the system. As a result, significant decreases in strength may occur before an appreciable change in vital capacity. In patients with generalized NMD, a 25% or greater decrease in vital capacity between the upright and supine postures has been shown to predict significant diaphragm muscle weakness. The weakened inspiratory pump has been associated with a stiffened chest wall and increased lung elastic loads, microatelectasis, reduced long volumes with restrictive pulmonary function impairment, CO 2 retention, and rapid, shallow breathing pattern. In addition, patients with NMD and respiratory muscle weakness may have a greater susceptibility to fatigue.
Weakness of the respiratory pump can be measured as a decrease in its pressure-generating capacity. In many NMDs the strength of the respiratory pump is poorly correlated with that of the limb muscles, and should be evaluated separately. Volitional pressure generation can be expressed as the maximal transdiaphragmatic pressure, described later.
Maximal Inspiratory Pressure
Maximal inspiratory pressure (MIP) measures the global pressure capacity of the inspiratory muscle pump at the mouth or tracheostomy opening. It is a noninvasive test with established reference values for age and gender, in both children and adults. MIP can be influenced by lung volume and is typically measured from residual volume. Accurate measurements may also be challenging for patients who are mechanically ventilated or have difficulty consistently following commands. In these cases, valid maximal efforts can be obtained with short bouts of inspiratory occlusions.
Nasal Sniff Pressure
Alternatively, nasal sniff pressures mimic a natural motor function, and maximal sniff maneuvers correlate well with MIP measurements at both the mouth and esophagus. In patients with NMD and severe ventilatory weakness, sniff pressure correlates well with restrictions in vital capacity related to pulmonary restrictive disease and/or progression of diaphragm dysfunction. Whereas MIP is a static maneuver, sniff is quasi-isometric, and different inspiratory muscle recruitment patterns have been noted during each maneuver. Thus, the combined use of MIP and maximal sniff pressures may prove valuable for accurate diagnosis of weakness and longitudinal follow-up of patients with NMD.
Transdiaphragmatic Pressure
The presence of specific diaphragm muscle weakness can be determined by the transdiaphragmatic pressure (Pdi) gradient. Balloon catheters or microtransducers placed in the esophagus (Pes) and stomach (Pga) can be used to estimate abdominal and pleural pressures. In healthy individuals, Pes becomes more negative during inspiration whereas Pga becomes more positive. Pdi can be evaluated during maximal voluntary inspiratory maneuvers or during contractions evoked by electrical or magnetic stimulation. In addition, Pdi during tidal breathing and inspiratory challenges may identify the fatigability of the diaphragm muscle. The use of esophageal manometry can provide a greater insight into specific diaphragm muscle weakness but the procedure is invasive, and catheter placement may be poorly tolerated by patients.
Diaphragmatic paralysis results in an upward movement of the diaphragm, resulting in a negative Pga during inspiration. As a result, the Pdi approaches zero. This process is accompanied by asynchronous, paradoxic breathing. The timing and extent of the chest and abdominal wall movements can be quantified during tidal breathing, as well as maximal efforts, by respiratory inductance plethysmography.
Evoked Phrenic Motor Unit Function
Nonvolitional assessments of diaphragm contractile function reduce the variability of voluntary, maximal-effort maneuvers and remove the role of patient cooperation from the examination. Diaphragm contractions can be elicited by supramaximal electrical or magnetic stimulation of the phrenic nerves and be measured by Pdi or electromyogram (EMG) responses. In patients with DMD and severe diaphragm dysfunction, diaphragm electrophysiological responses remained present even when Pdi responses to phrenic stimulation could not be detected. The diaphragm motor responses revealed a normal to elevated ventilatory drive despite an undetectable Pdi. In conjunction with needle EMG activity at rest and during graded contractions, the electrophysiological features elicited during phrenic stimulation can differentiate patients with an isolated neural pathology from those with a purely muscular impairment or a mixed pathology.
Inspiratory muscle fatigue
Inspiratory muscle weakness yields a progressive reduction in expansion of lung and chest wall during inhalation, resulting in a gradually reduced compliance and relative increase in the mechanical load during breathing. An imbalance between breathing loads and the pressure-generating capacity of the respiratory pump can lead to fatigue and ventilatory failure. The pressure-time index (PTI) can estimate the threshold for diaphragm fatigue. The PTI consists of two components: the relative pressure required of the diaphragm to generate a tidal breath and the portion of the respiratory cycle spent in inspiration. In healthy individuals, the fatigue threshold is a PTI in excess of 0.15. When relative breathing loads increase, patients with neuromuscular weakness may shorten inspiratory time and lower Pdi to minimize the work of breathing. However, this strategy of rapid, shallow breathing has been associated with an increased elastic load of the lung and hypercapnia. It has been shown that the PTI threshold for fatigue may be closer to 0.10 to 0.12 for patients with quadriplegia, and a low fatigue threshold is also thought to apply to patients with chronic NMD.
Expiratory muscle dysfunction
Expiratory muscle weakness results in a reduced ability to lower volume below functional residual capacity, resulting in declines of expiratory reserve volume and residual volume. Patients become less capable of producing the intrathoracic pressures necessary to generate effective cough flows and clear lung secretions. An inability to clear airway secretions is a serious problem in neuromuscular dysfunction, and is associated with an increased risk of mortality. Maximal expiratory pressure (MEP) is a noninvasive estimate of expiratory muscle strength, typically measured from total lung capacity (TLC). MEP is the expiratory equivalent of MIP, and the advantages and limitations of the MEP maneuver are similar to those of MIP. Although MEP decreases with the progression of NMD, it has not been found to be an independent predictor of hypercapnia. MEP is a significant predictor of peak cough flow in patients with DMD and ALS. Cough gastric pressure has been found to correlate well with MEP and may better differentiate weakness from technical difficulties when MEP is reduced.
Congenital NMD with Severe Respiratory Dysfunction: A Description of XLMTM in Humans
XLMTM, classified as a congenital myopathy, is an inherited disease transmitted on the X chromosome. It is characterized by marked muscle weakness, hypotonia, and feeding and breathing difficulties in male infants. Many XLMTM patients die during their first year of life, due to respiratory insufficiency. Affected infants may also present at birth with hypotonia, external ophthalmoplegia, and macrosomia. Female carriers do not display symptoms of the disease unless affected with other X-linked disorders. The incidence of XLMTM in humans is estimated to be 1 in 50,000 male births. Patients who survive past birth often have prolonged ventilator dependence and delayed motor milestones, but usually have intact intelligence. Long-term survivors of the disease (>1 year), appear to be at risk for medical complications involving other organ systems in addition to complications derived from being partially or completely ventilator dependent.
Diagnosis of XLMTM typically follows a muscle biopsy in which characteristic muscle histopathology has been identified as small, rounded muscle fibers with centrally located nuclei surrounded by a halo devoid of contractile elements and sarcoplasmic disorganization, reminiscent of the fetal stage of skeletal muscle, hence the name myotubular myopathy. The muscle fibers contain abnormal mitochondria, and type I fiber hypotrophy is present in patients afflicted with XLMTM. This histopathology does not appear to change significantly during the course of the disease, despite clinical deterioration.
Animal Models of XLMTM
To more fully define the progression and understanding of the mechanism and pathogenesis of human disease, a well-defined animal model is necessary. Animal models also allow for the development of therapeutic strategies that more accurately reflect disease severity and immunologic problems. A relevant animal model, and therefore species used, would be one in which the biological response to therapy would be expected to mimic the human response. Safety and efficacy data are needed prior to entry of any therapeutic agents into clinical trials. Some animal models of NMD appear as true counterparts to human disorders; others resemble but do not duplicate disease in humans. Still others require further study to determine the degree of compatibility.
Zebrafish and murine models of XLMTM have been recently established. The discovery, further classification, and additional testing of a recently discovered canine model of XLMTM has broadened the field for this disease. These models have played an important role in understanding the pathogenesis of how loss of myotubularin leads to respiratory weakness and death. Together these various species with myotubularin deficiency can be used to demonstrate different aspects of muscle maturation, maintenance, growth, and development. Findings in animals can lead to direct clinical translation, application, and use and refinement of diagnostic and therapeutic modalities for XLMTM patients. Tables 1–3 compare and contrast the existing models of XLMTM.
| Zebrafish | Mouse | Mouse | Canine | |
|---|---|---|---|---|
| Morpholino Knockdown | Myotubularin-Deficient KO ( Mtm1 KO) | HSA Mutant | ||
| Descriptor | Genetically modified | Genetically modified | Genetically modified | Naturally occurring, spontaneous |
| Gene loci | Not available | MTM1 | Deletion of MTM1 exon 4 | MTM1 |
| Mutation | Antisense morpholino used to interrupt MTM1 transcription | Frameshift mutation | Missense variation | |
| Expression | Both males and females | Sex linked: seen in the hemizygous male | Sex linked: seen in the hemizygous male | Sex linked: seen in the hemizygous male |
| Phenotype | Rapidly fatal | Rapidly fatal | Severe and rapidly fatal | Progressive |
| Screening | Phenotypic appearance | Tail snip | Cheek swab | |
| Genetic manipulability | Functional gene knockdown |
| Zebrafish | Mouse | Mouse | Canine | |
|---|---|---|---|---|
| Clinical features |
|
|
|
|
| Histopathologic features |
|
|
|
|
| Respiratory function | Not tested | Paradoxic breathing |
| Zebrafish | Mouse | Canine | |
|---|---|---|---|
| Average litter size | Large | 6–9 | 7 |
| Affected animal average life span | 7–9 wk/6–14 wk | 14–19 wk | |
| Time to detection of disease | Within 48–72 h postfertilization | 4 wk | 9–10 wk |
| Reproducibility | High | High | Dependent on Mendelian genetics |
| Muscle differentiation | At primary myogenesis | Complete at birth | 4 wk postpartum |
| Appropriate use |
|
|
|
Appropriate use of each species can be guided by several preclinical considerations. For example, the small size of mice guarantees that reagents can be produced in sufficient quantity and facilitates delivery of reagent in high doses. However, there are other aspects that may be more accurately performed in a larger model because of physiology, therapeutic feasibility and monitoring, and toxicity. If delivering therapeutic agents, large volumes may be required for translation to human medicine, which becomes impractical in mice. The larger canine model allows for studies of safety and efficacy to be performed, and dose-escalation studies are more easily executed. Specifically, this becomes important when considering intramuscular injections of gene-therapy vectors. For example, direct injection of plasmid DNA into muscles for gene therapy is more practical, feasible, and plausible when using the canine model as opposed to the murine model of XLMTM, and this holds especially true for respiratory muscle injections to test for therapeutic efficiency. The ability to provide additional medical and nutritional support to affected dogs as opposed to mice may allow the model to survive longer periods of time, thus allowing long-term evaluation to establish the typical chronologic sequence of clinical and histologic signs for XLMTM. Box 1 describes in further detail the relative advantages and disadvantages of the canine model of XLMTM.
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