Pompe disease is a severe form of muscular dystrophy due to glycogen accumulation in all tissues, especially striated muscle. Disease severity is directly related to the deficiency of acid α-glucosidase (GAA), which degrades glycogen in the lysosome. Respiratory dysfunction is a hallmark of the disease, muscle weakness has been viewed as the underlying cause, and the possibility of an associated neural contribution has not been evaluated previously. Therefore, we examined behavioral and neurophysiological aspects of breathing in 2 animal models of Pompe disease—the Gaa ^−/− mouse and a transgenic line (MTP) expressing GAA only in skeletal muscle, as well as a detailed analysis of the CNS in a Pompe disease patient. Glycogen content was elevated in the Gaa ^−/− mouse cervical spinal cord. Retrograde labeling of phrenic motoneurons showed significantly greater soma size in Gaa ^−/− mice vs. isogenic controls, and glycogen was observed in Gaa ^−/− phrenic motoneurons. Ventilation, assessed via plethysmography, was attenuated during quiet breathing and hypercapnic challenge in Gaa ^−/− mice (6 to >21 months of age) vs. controls. We confirmed that MTP mice had normal diaphragmatic contractile properties; however, MTP mice had ventilation similar to the Gaa ^−/− mice during quiet breathing. Neurophysiological recordings indicated that efferent phrenic nerve inspiratory burst amplitudes were substantially lower in Gaa ^−/− and MTP mice vs. controls. In human samples, we demonstrated similar pathology in the cervical spinal cord and greater accumulation of glycogen in spinal cord compared with brain. We conclude that neural output to the diaphragm is deficient in Gaa ^−/− mice, and therapies targeting muscle alone may be ineffective in Pompe disease.

Pompe disease is caused by a lack of functional lysosomal acid -glucosidase (GAA) and can ultimately lead to fatal hypertrophic cardiomyopathy and respiratory insufficiency. Previously, we demonstrated the ability of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to restore the therapeutic levels of cardiac and diaphragmatic GAA enzymatic activity in vivo in a mouse model of Pompe disease. We have further characterized cardiac and respiratory function in rAAV2/1-treated animals 1 year post-treatment. Similar to the patient population, electrocardiogram measurements (P-R interval) are significantly shortened in the Pompe mouse model. In rAAV2/1-treated mice, we show a significant improvement in cardiac conductance with prolonged P-R intervals of 39.341.6 ms, as compared to untreated controls (35.580.57 ms) (P0.05). In addition, we note a significant decrease in cardiac left ventricular mass from 181.9910.70 mg in untreated controls to 141.9719.15 mg in the rAAV2/1-treated mice. Furthermore, the mice displayed an increased diaphragmatic contractile force of approximately 90% of wild-type peak forces with corresponding improved ventilation (particularly in frequency, minute ventilation, and peak inspiratory flow). These results demonstrate that in addition to biochemical and histological correction, rAAV2/1 vectors can mediate sustained physiological correction of both cardiac and respiratory function in a model of fatal cardiomyopathy and muscular dystrophy.

Pompe disease is a muscular dystrophy that results in respiratory insufficiency. We characterized the outcomes of targeted delivery of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to diaphragms of Pompe mice with varying stages of disease progression. We observed significant improvement in diaphragm contractile strength in mice treated at 3 months of age that is sustained at least for 1 year and enhanced contractile strength in mice treated at 9 and 21 months of age, measured 3 months post-treatment. Ventilatory parameters including tidal volume/inspiratory time ratio, minute ventilation/expired CO₂ ratio, and peak inspiratory airflow were significantly improved in mice treated at 3 months and tested at 6 months. Despite early improvement, mice treated at 3 months and tested at 1 year had diminished normoxic ventilation, potentially due to attenuation of correction over time or progressive degeneration of nontargeted accessory tissues. However, for all rAAV2/1-treated mice (treated at 3, 9, and 21 months, assayed 3 months later; treated at 3 months, assayed at 1 year), minute ventilation and peak inspiratory flows were significantly improved during respiratory challenge. These results demonstrate that gel-mediated delivery of rAAV2/1 vectors can significantly augment ventilatory function at initial and late phases of disease in a model of muscular dystrophy.

The flipped classroom was utilized in a two-semester, high-content science course that enrolled between 50 and 80 students at a small liberal arts college. With the flipped model, students watched ~20-min lectures 2 days/wk outside of class. These videos were recorded via screen capture and included a detailed note outline, PowerPoint slides, and review questions. The traditional format included the same materials, except that lectures were delivered in class each week and spanned the entire period. During the flipped course, the instructor reviewed common misconceptions and asked questions requiring higher-order thinking, and five graded case studies were performed each semester. To determine whether assessments included additional higher-order thinking skills in the flipped vs. traditional model, questions across course formats were compared via Blooms Taxonomy. Application-level questions that required prediction of an outcome in a new scenario comprised 38 ± 3 vs. 12 ± 1% of summative assessment questions (<0.01): flipped vs. traditional. Final letter grades in both formats of the course were compared with major GPA. Students in the flipped model performed better than their GPA predicted, as 85.5% earned a higher grade (vs. 42.2% in the traditional classroom) compared with their major GPA. These data demonstrate that assessments transitioned to more application-level compared with factual knowledge-based questions with this particular flipped model, and students performed better in their final letter grade compared with the traditional lecture format. Although the benefits to a flipped classroom are highlighted, student evaluations did suffer. More detailed studies comparing the traditional and flipped formats are warranted.

Increased free radical production and oxidative damage in ageing muscle may be a contributing factor to the development of sarcopenia. It has been suggested that the accumulation of iron may be an underlying factor in the development of oxidative stress in ageing tissues, including skeletal muscle. At present, however, the mechanisms responsible for ageing‐associated muscle iron accumulation are unknown. These experiments tested the hypothesis that ageing‐associated elevations in skeletal muscle iron are accompanied by altered expression of key regulators of intracellular iron status. We determined non‐haem iron, oxidative injury, and expression levels of iron regulation proteins in plantaris muscles harvested from 6‐ and 24‐ to 26‐month‐old Fisher 344 rats (n= 10 per group). Ageing resulted in a 62% elevation in skeletal muscle non‐haem iron (P < 0.05) and higher protein oxidative damage (P < 0.05). Notably, ageing was associated with elevated expression of ferritin (heavy chain, +56.2‐fold; light chain, +7.3‐fold), an important iron storage protein. Conversely, the iron transport protein transferrin receptor‐1 demonstrated dramatic downregulation (−10.8‐fold; P < 0.05) in old muscle, whereas the level of divalent metal transporter‐1 protein expression was unaltered. No change in protein level of iron regulatory protein‐1 was observed. In summary, these results demonstrate the occurrence of altered iron regulation concomitant with iron accumulation and oxidative stress in aged skeletal muscle. Importantly, the maintenance of divalent metal transporter‐1 protein expression into old age could play a role in the accumulation of skeletal muscle iron.