IntroductionThe 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.MethodsNeonatal 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.ResultsWe 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.DiscussionWe 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.

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.

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. TrkBF616A 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.

Ultrasound-Based Interprofessional Education: Priming Students for Interprofessional Collaboration Charlene M. Gaw, MD, MPH, Christopher M. Gibbs, MD; Jennifer A. Knight, MD; Nathan J. Hellyer, PT, PhD; Natalie R. Langley, PhD; Sonya E. Van Nuland, PhD Full Text: PDF DOI: 10.15640/ijhs.v8n3a1

PURPOSE::Heart rate variability (HRV), or the beat-to-beat variance in heart rate, is an adjunct measure of stress and physiological fatigue. Physically active individuals and athletes may use HRV as a measure of recovery from physical exhaustion, but change in HRV naturally fluctuates and meaningful change has not been well described. Therefore, the purpose of our investigation is to investigate reliability and minimal detectable difference in HRV measurement. METHODS::We employed a test-retest reliability design with five minute resting heart rate measurements taken one week apart in eleven male and eleven female subjects (23±1 years old; BMI 22.7±2.3 kg/m2) positioned in a supine posture. HRV was collected by a Polar H7 heart rate sensor strap with data collected by a commercially available HRV android application, HRV Elite. Artifact detection and HRV analysis was performed using ARTiiFACT software to generate the root mean square of the successive differences (RMSSD) values for each HRV measurement. Intraclass correlation coefficients (ICC) were calculated to examine reliability and a minimal detectable difference was calculated to examine change detectable beyond the limitations of error. RESULTS: RMSSD means on trial one and two were 75.0 (s.d.=27.8) milliseconds and 68.3 (s.d.=28.8) milliseconds, respectively. We observed an ICC of 0.947 (95% confidence interval 0.803-0.987). We calculated the minimal detectable difference to be 18.4 milliseconds. CONCLUSIONS: HRV measurements taken from a heart rate strap and android phone application appear reliable in young, healthy subjects at rest as indicated by a relatively high ICC. However, a daily change in RMSSD needs to be considered with respect to innate measurement error in order to reflect meaningful change, which for the Polar H7 sensor and HRV Elite application pairing appears to be slightly greater than eighteen seconds.

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 InformationDepartment of Anatomy, Mayo Clinic College of Medicine and Science, Mayo Clinic, Rochester, MN; Social Science and Humanities Research Council, Government of Canada

BACKGROUND:Assessment of diaphragm contraction may be useful for identifying impairments in patients with movement dysfunction involving trunk stabilization, respiration, or both. Real-time ultrasound imaging is a readily available technology that can be used to quickly assess this aspect of diaphragm activity. Although previous studies have examined diaphragm contraction in the supine posture, a comparison of measurements between supine and upright postures has not been made.OBJECTIVE:To examine whether diaphragm thickness measurements differ among 3 different body postures in healthy subjects.DESIGN:Descriptive repeated measures.SETTING:Clinical laboratory.PATIENTS (OR PARTICIPANTS):Twenty-four healthy subjects (12 male and 12 female) aged 22-35 years old were recruited and completed the study.METHOD:Diaphragm thickness was assessed in via B-mode ultrasound imaging in supine, seated, and standing postures. Measurements of diaphragm thickness were taken in the zone of apposition during maximal inspiration to total lung capacity (TLC) and end-tidal expiratory lung volume (EELV). A thickness ratio (inspiration thickness/expiration thickness) was calculated to compare relative diaphragm contraction during each condition.MAIN OUTCOME MEASUREMENTS:The primary dependent variable was diaphragm thickness (mm).RESULTS:Average diaphragm thickness at EELV and maximum TLC were more than 20% greater in the seated and standing postures than in supine (P < .05). Moreover, the diaphragm was approximately 205% thicker at TLC than at EELV (P < .05). Relative inspiratory to expiratory thickness ratios (TLC/EELV) did not differ among postures (P = .24).CONCLUSIONS:The diaphragm is thicker when the body is in more upright postures (standing and sitting versus supine) perhaps due to greater vertical gravitational load on the muscle and associated change in the resting length of the muscle fibers. Thus it appears that ultrasound imaging may be a sensitive tool to examine changes in diaphragm contraction during varying postural tasks.LEVEL OF EVIDENCE:IV.

Hellyer, NJ, Folsom, IA, Gaz, DV, Kakuk, AC, Mack, JL, and Ver Mulm, JA. Respiratory muscle activity during simultaneous stationary cycling and inspiratory muscle training. J Strength Cond Res 29(12): 3517-3522, 2015Inspiratory muscle training (IMT) strengthens the muscles of respiration, improves breathing efficiency, and increases fitness. The IMT is generally performed independently of aerobic exercise; however, it is not clear whether there is added benefit of performing the IMT while simultaneously performing aerobic exercise in terms of activating and strengthening inspiratory muscles. The purpose of our study was to determine the effect of IMT on respiratory muscle electromyography (EMG) activity during stationary cycling in the upright and drops postures as compared with that when the IMT was performed alone. Diaphragm and sternocleidomastoid EMG activity was measured under different resting and cycling postures, with and without the use of the IMT at 40% maximal inspiratory pressure (n = 10; mean age 37). Cycling in an upright posture while simultaneously performing the IMT resulted in a significantly greater diaphragm EMG activity than while performing the IMT at rest in upright or drops postures (p 0.05). Cycling in drops postures while performing the IMT had a significantly greater diaphragm EMG activity than when performing the IMT at rest in either upright or drops postures (p 0.05). Sternocleidomastoid muscle activity increased with both cycling and IMT, although posture had little effect. These results support our hypothesis in that the IMT while cycling increases respiratory EMG activity to a significantly greater extent than when performing the IMT solely at rest, suggesting that the combination of IMT and cycling may provide an additive training effect.

Medicine is increasingly focused on team-based practice as interprofessional cooperation leads to better patient care. Thus, it is necessary to teach teamwork and collaboration with other health care professionals in undergraduate medical education to ensure that trainees entering the workforce are prepared to work in teams. Gross anatomy provides an opportunity to expose students to interprofessional education (IPE) early in their training. The purpose of this study is to describe an IPE experience and report if the experience has lasting influence on the participating students. The Readiness for Interprofessional Learning Scale (RIPLS) questionnaire was administered to first year medical (MD) and physical therapy (PT) students at Mayo Medical School and Mayo School of Health Sciences. Results demonstrated an openness on the part of the students to IPE. Interprofessional education experiences were incorporated into gross anatomy courses in both medical and PT curricula. The IPE experiences included a social event, peer-teaching, and collaborative clinical problem-solving sessions. These sessions enhanced gross anatomy education by reinforcing previous material and providing the opportunity to work on clinical cases from the perspective of two healthcare disciplines. After course completion, students again completed the RIPLS. Finally, one year after course completion, students were asked to provide feedback on their experience. The post-curricular RIPLS, similar to the pre-curricular RIPLS, illustrated openness to IPE from both MD and PT students. There were however, significant differences in MD and PT perceptions of roles and responsibilities. One-year follow-up indicated long-term retention of lessons learned during IPE.

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.

Medical professionals and public consumers expect that new physical therapy graduates possess cognitive, technical, and behavioral skills required to provide safe and high-quality care to patients. The purpose of this study was to determine if a repertoire of ten professional behaviors assessed at the beginning of doctorate of physical therapy education and before the first significant clinical internship could be enhanced in a semester course in gross human anatomy using individual formative feedback. During the human anatomy course, 28 first-year physical therapy students completed six biweekly, anonymous self- and peer assessment surveys that targeted ten professional behaviors important to physical therapists. All professional behaviors were assessed using a five-point Likert scale. Feedback reports occurred at week eight (mid-semester) and week 16 (end-of-semester) and comprised the direct intervention components of this study. At the midpoint of the semester, professional behavior scores and narrative comments from weeks two, four, and six were compiled and shared with each student by one of three faculty members in a feedback session. Students then submitted biweekly self-and peer professional behavior assessments (weeks 10, 12, and 14) for the remainder of the human anatomy course. Differences between preintervention and postintervention scores for each of the ten professional behaviors were compared using the Wilcoxon signed-ranks test. Upon receiving mid-semester individual feedback, students demonstrated significant improvement in each of the ten professional behaviors. Results from this study indicated a gross anatomy laboratory dissection experience during the first academic semester provided an effective opportunity for teaching and assessing professional behaviors of doctoral students in physical therapy.

STUDY DESIGN Clinical measurement, cross-sectional. OBJECTIVES To establish a set of normal values for diaphragm thickening with tidal breathing in healthy subjects. BACKGROUND Normal values for diaphragm contractility, as imaged sonographically, have not been described, despite the known role of the diaphragm in contributing to spinal stability. If the normal range of diaphragm contractility can be defined in a reliable manner, ultrasound has the potential to be used clinically and in research as a biofeedback tool to enhance diaphragm activation/contractility. METHODS B-mode ultrasound was performed on 150 healthy subjects to visualize and measure hemi-diaphragm thickness on each side at resting inspiration and expiration. Primary outcome measures were hemi-diaphragm thickness and thickening ratio, stratified for age, gender, and body mass index. Interrater and intrarater reliability were also measured. RESULTS Normal thickness of the diaphragm at rest ranged from 0.12 to 1.18 cm, with slightly greater thickness in men but no effect of age. Average ± SD change in thickness from resting expiration to resting inspiration was 20.0% ± 15.5% on the right and 23.5% ± 24.4% on the left; however, almost one third of healthy subjects had no to minimal diaphragm thickening with tidal breathing. CONCLUSION There is wide variability in the degree of diaphragm contractility during quiet breathing. B-mode ultrasound appears to be a reliable means of determining the contractility of the diaphragm, an important muscle in spinal stability. Further studies are needed to validate this imaging modality as a clinical tool in the neuromuscular re-education of the diaphragm to improve spinal stability in both healthy subjects and in patients with low back pain.

Hellyer, NJ, Nokleby, JJ, Thicke, BM, Zhan, W-Z, Sieck, GC, and Mantilla, CB. Reduced ribosomal protein S6 phosphorylation after progressive resistance exercise in growing adolescent rats. J Strength Cond Res 26(6): 1657-1666, 2012-The purpose of this study was to investigate moderate intensity progressive resistance exercise (PRE) in growing adolescent rats and its effect on muscle hypertrophy (defined as an increase in fiber cross-sectional area [CSA]). We hypothesized that in adolescent animals moderate intensity PRE would increase (a) fiber CSA; (b) myosin heavy chain (MyHC) content; and (c) expression and phosphorylation of cell signaling molecules involved in translational regulation, compared with that in age-matched sedentary (SED) controls. In the PRE group, 3-week-old male rats were trained to climb a vertical ladder as a mode of PRE training such that by 10 weeks all animals in the PRE group had progressed to carry an additional 80% of their body weight per climb. In agreement with our hypotheses, we observed that 10 weeks of moderate PRE in adolescent animals was sufficient to increase the CSA of muscle fibers and increase MyHC content. The average muscle fiber CSA increased by >10%, and the total MyHC content increased by 35% (p<0.05) in the PRE group compared with that in the SED animals. Concurrently, we investigated sustained changes in the expression and phosphorylation of key signaling molecules that are previously identified regulators of hypertrophy in adult animal models. Contrary to our hypotheses, expression and phosphorylation of the translational regulators mammalian target of rapamycin and Akt were not increased in the PRE group. In addition, we observed that the ratio of phosphorylated-to-unphosphorylated ribosomal protein S6 (rpS6) was reduced over sixfold in PRE animals (p<0.05) and that total rpS6 protein levels were unchanged between PRE and SED animals (p<0.05). We conclude that moderate intensity PRE is sufficient to induce muscle hypertrophy in adolescent animals, whereas the signaling mechanisms associated with muscle hypertrophy may differ between growing adolescents and adults.

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.

The action of muscles about joints can be explained through analysis of their spatial relationship. A functional model of these relationships can be valuable in learning and understanding the muscular action about a joint. A model can be particularly helpful when examining complex actions across multiple joints such as in the digital extensor mechanism of the hand. A functional model using two hair bands representing the components of the digital extensor mechanism is described. Through superimposition of the model over one's digit, the combined actions of metacarpophalangeal joint flexion and interphalangeal joint extension are clearly demonstrated. The model provides a conceptual demonstration to enhance understanding of this unique combination of muscle actions about multiple joints.

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.

Previous studies showed that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) results in loss of myosin heavy chain protein by 1 day after DNV. We hypothesize that DNV decreases net protein balance as a result of activation of the ubiquitin-proteasome pathway. In DIAm strips, protein synthesis was measured by incorporation of 3H-Tyr, and protein degradation was measured by Tyr release at 1, 3, 5, 7, and 14 days after DNV. Total protein ubiquitination, caspase-3 expression/activity, and actin fragmentation were analyzed by Western analysis. We found that, at 3 days after DNV, protein synthesis increased by 77% relative to sham controls. Protein synthesis remained elevated at 5 (85%), 7 (53%), and 14 days (123%) after DNV. At 5 days after DNV, protein degradation increased by 43% relative to sham controls and remained elevated at 7 (49%) and 14 days (74%) after DNV. Thus, by 5 days after DNV, net protein balance decreased by 43% compared with sham controls and was decreased compared with sham at 7 (49%) and 14 days (72%) after DNV. Protein ubiquitination increased at 5 days after DNV and remained elevated. DNV had no effect on caspase-3 activity or actin fragmentation, suggesting that the ubiquitin-proteasome pathway rather than caspase-3 activation is important in the DIAm response to DNV. Early loss of contractile proteins, such as myosin heavy chain, is likely the result of selective protein degradation rather than generalized protein breakdown. Future studies should evaluate this selective effect of DNV.