Myotonic Dystrophy Transgenic Mice Exhibit Pathologic Abnormalities in Diaphragm Neuromuscular Junctions and Phrenic Nerves

Abstract

Myotonic dystrophy Type 1 (DM-1) is caused by abnormal expansion of a (CTG) repeat located in the DM protein kinase gene. Respiratory problems have long been recognized to be a major feature of this disorder. Because respiratory failure can be associated with dysfunction of phrenic nerves and diaphragm muscle, we examined the diaphragm and respiratory neural network in transgenic mice carrying the human genomic DM-1 region with expanded repeats of more than 300 CTG, a valid model of the human disease. Morphologic and morphometric analyses revealed distal denervation of diaphragm neuromuscular junctions in DM-1 transgenic mice indicated by a decrease in the size and shape complexity of end-plates and a reduction in the concentration of acetyl choline receptors on the postsynaptic membrane. More importantly, there was a significant reduction in numbers of unmyelinated, but not of myelinated, fibers in DM-1 phrenic nerves; no morphologic alternations of the nerves or loss of neuronal cells were detected in medullary respiratory centers or cervical phrenic motor neurons. Because neuromuscular junctions are involved in action potential transmission and the afferent phrenic unmyelinated fibers control the inspiratory activity, our results suggest that the respiratory impairment associated with DM-1 may be partially due to pathologic alterations in neuromuscular junctions and phrenic nerves.

Functional and histopathological identification of the respiratory failure in a DMSXL transgenic mouse model of Myotonic Dystrophy

“…Moreover, the AChR density on postsynaptic membranes was significantly lower. It should be noted that in previous experiments in DM1 transgenic mice carrying only 500 CTG repeats we detected similar abnormalities in the structure of diaphragmatic EPs (Panaite et al, 2008). The fact that the statistical analysis revealed that the pathological changes in diaphragmatic EPs found in DMSXL mice, carrying about 1300 CTG repeats, do not vary significantly from those observed in DM1 mice indicates that there is no relationship between the severity of EP alterations and length of the CTG repeat.…”

“…After testing the respiratory function, mice were deeply anesthetized and the brainstem, cervical spinal cord, right and left phrenic nerves and diaphragm muscles carefully removed from each animal as previously described (Panaite et al, 2008; Panaite et al, 2011). …”

“…The percentage of denervated endplates (EPs) was estimated and the morphometric parameters (area, shape complexity and fluorescence intensity of rhodamine-α-BTX labeling) of each EP were measured and calculated as described in our previous studies (Panaite et al, 2008; Panaite et al, 2011). More than 1500 EPs were measured from each mouse line ( n =7 DMSXL and control mice).…”

“…For electron microscopy examination, ultrathin sections (80 nm) were cut and contrasted with uranyl acetate and lead citrate. Sections were viewed in the Zeiss EM 10C electron microscope and morphometric analysis was performed as previously described (Gantelet et al, 2007; Panaite et al, 2008). The number of myelinated and unmyelinated axons was counted and the thickness of the myelin sheath calculated ( n =7 for each DMSXL and controls).…”

Splicing biomarkers of disease severity in myotonic dystrophy

“…It is also possible that muscle deficits are not cell autonomous, but depend partly on RNA toxicity in other cells, such as motor neurons. 43, 44 A limitation of our study, however, is that resolution and sizing of large CTG fragments on Southern blots is not precise. Alternative technologies to confirm expansion size are needed but are not currently available.…”