Hellyer, Nathan J.

Introduction The nerve-derived growth factor neuregulin (NRG) plays a role in the regulation of skeletal muscle mass through Akt and mTOR signaling transduction pathways that regulate both protein synthesis and degradation. We previously reported that NRG increases muscle protein synthesis (~20%) in a PI3 kinase (PI3K)/Akt-dependent manner. However, the effects of NRG on protein degradation are still poorly understood and are needed to elucidate the role of NRG in the maintenance of skeletal muscle protein balance. Methods Neonatal diaphragm muscle ex vivo preparations were pharmacologically treated with NRG and pharmacological inhibitors of PI3K (LY294002, 50 μM), MEK (PD98059, 50 μM) or mTOR (rapamycin, 100 nM). Tyrosine release from muscle was used as a surrogate measure of protein degradation. Results We report that basal protein degradation in the neonatal rat diaphragm muscle is significantly reduced by NRG treatment (19%). Basal protein degradation was increased following treatment with inhibitors of PI3K, MEK or mTOR, with inhibition of each pathway sufficient to increase basal protein degradation greater than 30%. Importantly, NRG treatment in the presence of each of these inhibitors blunts the increase in protein degradation induced by inhibition of PI3K, MEK or mTOR. NRG effects were significantly blunted by rapamycin (p < 0.05 compared to NRG alone), but not by LY294002 or PD98059. Discussion We suggest that both the PI3K/Akt and MAP kinase pathways are important for NRG effects on protein degradation, but that mTOR may be a critical modulator of these effects and thus of protein balance in skeletal muscle.

Ultrasound has been used more frequently in the anatomical sciences to reinforce core anatomy concepts in the classroom and introduce commonly applied imaging technologies encountered in a clinical setting. Interprofessional Education (IPE) is being incorporated into health sciences training as a learning model for real world interactions. The primary objective of the current study was to determine the impact of an IPE ultrasound curriculum on students' anatomy and ultrasound knowledge, as well as attitudes towards interprofessional collaboration. We hypothesized that students who participated in an IPE experience would have improved recall of anatomical and ultrasound concepts as well as improved attitudes and perceptions towards IPE when compared to students who did not participate in an IPE session. First year medical students (MD, n = 40) and first year physical therapy students (PT, n = 25) were randomly assigned to PT‐only, MD‐only, or mixed PT‐MD ultrasound instruction groups studying the shoulder. Students were assessed using an anatomy and ultrasound knowledge test and University of the West of England Interprofessional Questionnaire (UWEIPQ) prior to and following the session. The results of this study suggest that while ultrasound instruction significantly improves ultrasound post‐test scores when compared to pre‐test scores regardless of group assignment (PT‐only: p < 0.001; MD‐only: p = 0.007; PT‐MD: p < 0.001), learning in an interprofessional PT‐MD group did not significantly impact knowledge recall of ultrasound concepts when compared to non‐IPE groups. Interestingly, students in PT‐MD groups had significantly higher anatomy post‐test scores when compared to the MD‐only groups (PT‐MD post‐test = 93.2%; MD‐only post‐test = 84.1%; p = 0.041), but not when compared to the PT‐only groups. Additionally, the results of the UWEIPQ are consistent with the impression that students who participate in an IPE experience are more likely to be cognizant of inherent biases in the interactions between different health professionals as well as demonstrate increased confidence in their interprofessional relationships. In conclusion, our study reinforces that IPE programs should quantify knowledge recall in order to assess the effectiveness of a specific program. Furthermore, such endeavors should assess important competencies including communication, teamwork, and interdisciplinary respect that are necessary to succeed in a multidisciplinary health care team. Support or Funding Information Department of Anatomy, Mayo Clinic College of Medicine and Science, Mayo Clinic, Rochester, MN; Social Science and Humanities Research Council, Government of Canada

Sarcopenia is the aging‐related loss of force‐generating capacity and atrophy of skeletal muscles and is a major contributor to morbidity and mortality in the elderly. The molecular mechanisms underlying sarcopenia remain to be elucidated. Skeletal muscles exhibit different rates of sarcopenia, generally reflecting their specific muscle fiber type composition. Muscles primarily composed of type IIx and/or IIb muscle fibers (i.e., extensor digitorum longus; EDLm) develop sarcopenia at a faster rate than other muscles. In the diaphragm, muscle fiber cross‐sectional area (CSA) is reduced in old age, predominantly in type IIx and/or IIb fibers. Autophagy is a multistep, catabolic process that targets dysfunctional cytoplasmatic structures including proteins and organelles for degradation and recycling. Autophagy is impaired in old age across several tissues, including motor neurons and skeletal muscle. Expression of core autophagy proteins can indicate changes in autophagy, e.g., LC3 reflects initiation/elongation steps and p62 clearance reflects degradation. We hypothesize that impaired autophagy is associated with the development of sarcopenia in type‐identified muscle fibers of the EDLm. Indeed, at 24‐months of age, the CSA of type I and IIa fibers was not reduced, whereas the CSA of type IIx and/or IIb fibers was ~25% smaller (p<0.01 in both cases), compared to 6‐month‐old mice. These findings are consistent with fiber type selective sarcopenia in the EDLm. Expression of LC3 and p62 in the EDLm of mice at 6‐ and 24‐ months of age was evaluated via immunofluorescence by quantification of the number of LC3 and p62 puncta per muscle fiber type. In type I and IIa fibers, the number of LC3 puncta increased by ~30% and of p62 increased three‐fold in 24‐ compared to 6‐month‐old mice (p<0.01 in both cases). In type IIx and/or IIb fibers, the number of LC3 puncta decreased by ~60% and p62 puncta increased by ~50% in 24‐ compared to 6‐month‐old mice (p<0.01 in both cases). Accordingly, compared to 6 month‐old mice, autophagic degradation is impaired across muscle fiber types in 24‐month old mice, but likely to a greater extent in type I and IIa fibers of old mice given the increase in both LC3 and p62. Autophagic initiation is primarily reduced in type IIx and/or IIb fibers in the EDL as indicated by the reduced LC3 expression. Taken together these findings indicate that fiber type selective sarcopenia may reflect differences in autophagy across muscle fiber types.

Neuregulin (NRG) is a member of the epidermal growth factor family, with 3 types varying in their structure and cellular expression but not in their receptor‐binding domain. NRG‐2 and NRG‐3 are primarily membrane bound, while NRG‐1 can be secreted. NRG‐1 is present in phrenic motor neurons. We previously reported that exogenous NRG containing only the receptor‐binding domain, increases protein synthesis in rat diaphragm. This effect was significantly inhibited by LY294002 indicating a PI 3‐kinase dependent mechanism. We also reported that NRG increases Akt phosphorylation. Interestingly, Akt phosphorylation is positively correlated with skeletal muscle growth and inversely correlated with atrophy. In this study, we hypothesized that NRG reduces protein degradation in rat diaphragm. Diaphragms were harvested from 4‐week old rats and incubated in the presence or absence of exogenous NRG. Protein degradation was measured by quantifying muscle tyrosine release via a fluorimetric assay. We found that NRG treatment resulted in a 50% reduction in tyrosine release from diaphragm preparations, indicating a reduction in basal protein degradation rates. These results suggest that phrenic motoneuron‐derived NRG‐1 regulates muscle protein synthesis and degradation rates, thereby playing a role in matching muscle fiber and motoneuron properties within motor units.

Neuregulin (NRG) is part of the epidermal growth factor family of proteins. Previous studies have shown that NRG activates PI3K/Akt pathways, leading to regulation of protein synthesis via downstream regulation of translation initiators. The principal translation initiator involved in protein synthesis is probably eIF4E, and the principal transcription factor regulating protein degradation is FOXO. We hypothesized that NRG signaling involves activation of the PI3K/Akt pathway, thus leading to an increase in expression of eIF4E and a decrease in expression of FOXO. An increase in eIF4E expression increases protein synthesis, and a decrease in FOXO expression reduces protein degradation. In this study we used dexamethasone, a synthetic glucocorticoid, to induce atrophy and cause an imbalance between protein synthesis and degradation in L6 myotubes. As expected, we found that after 48 hours of treatment with 10–100nM dexamethasone, eIF4E expression decreased and FOXO expression increased in a dose‐dependent manner. We found that treatment with 200 ng/ml NRG increased eIF4E expression. When L6 cells were treated with both NRG and dexamethasone for 48 hours, NRG blunted the catabolic effects of dexamethasone, thus increasing eIF4E expression and decreasing FOXO expression. These results indicate that NRG promotes protein synthesis and prevents protein degradation. Supported by NIH grant AR51173.

Unilateral denervation (DNV) of rat diaphragm muscle (DIAm) increases protein synthesis and degradation, with net protein breakdown by 14 days after DNV. We hypothesized that neural influences such as the nerve‐derived trophic factor neuregulin (NRG‐1) are essential in maintaining DIAm protein balance. NRG activates receptor tyrosine kinases of the ErbB family, with the ErbB2‐ErbB3 co‐receptor showing highest affinity for NRG‐1 and strong phosphoinositol‐3‐kinase (PI3K) activation. Both protein synthesis (via Akt phosphorylation) and degradation (via the transcription factor FoxO3A) are regulated by PI3K activity. We used Western blot analyses to assess NRG‐1, ErbB2, ErbB3 and FoxO3A protein expression at 1, 3, 7, and 14 days after DNV. By 1 day after DNV, NRG‐1 expression decreased, whereas ErbB2 and ErbB3 expression increased. Consistent with reduced NRG/ErbB signaling, association of the PI3K regulatory subunit p85 with ErbB3 decreased at 14 days after DNV. FoxO3A expression increased at 7 and 14 days after DNV, in agreement with delayed net protein breakdown. Importantly, DIAm inactivity induced by spinal hemisection had no effect on ErbB2, ErbB3, or FoxO3A expression. Taken together, these results indicate that DNV‐induced skeletal muscle adaptations result from removal of a trophic influence such as NRG‐1 rather than inactivity itself. Supported by NIH grant AR51173.

Muscle protein content reflects a balance between protein synthesis and degradation, which are likely regulated by neuromuscular activity and/or neurotrophin influence. In previous studies, we found that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) protein content after approximately 7 days. However, we did not determine whether the decrease in DIAm MHC protein content was the result of a decrease in protein synthesis, an increase in protein degradation, or both. We hypothesize that protein degradation plays a significant role in the decrease in DIAm protein content following DNV. In this study, we analyzed the ubiquitination state of cellular proteins as a marker of protein degradation. Conjugation of ubiquitin to cellular proteins targets proteins for degradation by proteosomes. We analyzed DIAm from adult male Sprague‐Dawley rats 3, 7 and 14 days after DNV. The DIAm was homogenized and protein ubiquitination was analyzed by Western analysis. No difference in ubiquitination was detected between control (CTL), sham surgery‐treated (sham), and 3‐day DNV animals. However, following 7 and 14 days of DNV, there was an approximate five‐fold increase in protein ubiquitination in DIAm compared to CTL and sham groups. These results demonstrate that DIAm protein ubiquitination increases between 3 and 7 days following DNV, and support the hypothesis that protein degradation contributes to the DNV‐induced decrease in DIAm MHC protein content. Supported by NIH grants AR51173 and HL37680.

Skeletal muscle growth is achieved by a net increase in muscle protein synthesis, decreased protein degradation or both. The Akt and mTOR signaling transduction pathways regulate both protein synthesis and degradation. We previously reported that the nerve‐derived growth factor neuregulin (NRG) increases muscle protein synthesis (~20%) in a PI3 kinase (PI3K)/Akt‐dependent manner. In the present study, we report that basal protein degradation in the diaphragm muscle is also significantly reduced by NRG treatment (23%). The NRG‐induced reduction of basal protein degradation is abrogated by pretreatment with inhibitors of PI3K (LY294002, 50 μM), mTOR (rapamycin, 100 nM) or MEK (PD98059, 50 μM). In fact, inhibition of any of these pathways alone is sufficient to increase basal protein degradation by greater than 30%. Importantly, NRG treatment nullifies the increase in protein degradation induced by PI3K or MEK inhibition, but not that induced by mTOR inhibition. Thus, unlike NRG effects on protein synthesis, the NRG‐induced decrease in protein degradation is abrogated not only by inhibition of the PI3K/Akt pathway, but also by inhibition of the MAP kinase. Based on these results, we suggest that both the PI3K/Akt and MAP kinase pathways are important for NRG effects on protein degradation, and that mTOR is a critical regulator of NRG effects on protein balance in skeletal muscle. Supported by NIH grant AR51173.

Interprofessional collaboration in healthcare is crucial for quality patient care. While it continues to be an expected competency, most educational programs lack interprofessional learning (IPL) experiences. We administered the five‐point Likert Readiness of Interprofessional Learning Scale (RIPLS) to first year medical (MD) and physical therapy (PT) students to evaluate attitudes toward IPL. Students agreed that “For small group learning to work, students need to trust and respect each other” (average of 4.6 out of five points). They also disagreed that learning with other healthcare students is a waste of time or unnecessary (averages of 1.91 and 1.98 respectively). Clearly students desire IPL experiences, and with this in mind, we established an IPL curriculum for their gross anatomy courses. Students were first grouped into interprofessional teams at a social event. In dissection lab, PT students taught upper limb and MD students taught thorax anatomy. We also conducted IPL anatomy based problem solving sessions in a classroom setting. Anatomy courses provide an ideal platform for IPL opportunities. Lab and classroom activities enable students to work together through a shared experience of understanding anatomical concepts from different perspectives. By establishing an IPL curriculum, we hoped to increase interprofessional collaboration early in students’ medical education. Our results indicate positive student attitude toward the learning experience.

Autophagy maintains cellular homeostasis by the elimination of damaged proteins or organelles. Our overarching hypothesis is that autophagy contributes to aging‐related neuromuscular dysfunction. The mechanisms regulating autophagy flux are promising therapeutic targets to mitigate the effects of neuromuscular dysfunction across conditions such as aging. In previous studies, we found that autophagy flux is reduced in early old age (18‐month old mice; 90% survival), with progressive autophagosome accumulation in old age evident by increased LC3 and p62 protein expression in cervical motor neurons (24‐month old mice; 75% survival). Neurotrophins acting through the tropomyosin‐related kinase receptor subtype B (TrkB) are critical to the maintenance of neuromuscular function including neuromuscular transmission in the diaphragm muscle and generation of maximal transdiaphragmatic pressure (Pdi), both in young and aged mice. Specifically, inhibition of TrkB signaling impairs maximal Pdi generation whereas activation of TrkB signaling blunts failure in neuromuscular transmission. We hypothesize that inhibiting TrkB kinase activity reduces autophagy flux resulting in increased LC3 and p62 expression, like the aging effects at phrenic motor neurons. TrkB F616A mice at 18‐ and 24‐ months of age, harboring a mutation that induces susceptibility to 1NMPP1‐mediated TrkB kinase inhibition, were randomized to receive 1‐week treatment with vehicle or 1NMPP1 (n=6 per age and treatment group). Protein expression of the autophagy markers LC3 and p62 in cervical spinal cord (CSC) segments C2‐C6 was analyzed by Western blotting. There was a significant effect on LC3 and p62 expression of age, but not of 1NMPP1 treatment, and no age*treatment interaction. LC3B expression increased by ~50% and p62 expression increased by ~30% in 24 month‐old compared to 18 month‐old mice. These results are consistent with previous reports that there is an aging‐related impairment of autophagy and accumulation of autophagosomes; however, inhibiting TrkB kinase activity does not modulate autophagy in these age groups. Understanding the role of TrkB signaling in mediating changes in autophagy and age‐related neuromuscular dysfunction will be important in advancing potential therapeutic applications of TrkB agonists.