Reduced exercise capacity occurs before intrinsic skeletal muscle dysfunction in experimental rat models of pulmonary hypertension.

Reduced exercise capacity in pulmonary hypertension (PH) significantly impacts quality of life. However, the cause of reduced exercise capacity in PH remains unclear. The objective of this study was to investigate whether intrinsic skeletal muscle changes are causative in reduced exercise capacity in PH using preclinical PH rat models with different PH severity. PH was induced in adult Sprague-Dawley (SD) or Fischer (CDF) rats with one dose of SU5416 (20 mg/kg) injection, followed by 3 weeks of hypoxia and additional 0-4 weeks of normoxia exposure. Control s rats were injected with vehicle and housed in normoxia. Echocardiography was performed to assess cardiac function. Exercise capacity was assessed by VO2 max. Skeletal muscle structural changes (atrophy, fiber type switching, and capillary density), mitochondrial function, isometric force, and fatigue profile were assessed. In SD rats, right ventricular systolic dysfunction is associated with reduced exercise capacity in PH rats at 7-week timepoint in comparison to control rats, while no changes were observed in skeletal muscle structure, mitochondrial function, isometric force, or fatigue profile. CDF rats at 4-week timepoint developed a more severe PH and, in addition to right ventricular dysfunction, the reduced exercise capacity in these rats is associated with skeletal muscle atrophy; however, mitochondrial function, isometric force, and fatigue profile in skeletal muscle remain unchanged. Our data suggest that cardiopulmonary impairments in PH are the primary cause of reduced exercise capacity, which occurs before intrinsic skeletal muscle dysfunction.

An emerging field: An evaluation of biomedical graduate student and postdoctoral education and training research across seven decades.

Biomedical graduate student and postdoctoral education and training research has expanded greatly over the last seven decades, leading to increased publications and the emergence of a field. The goal of this study was to analyze this growth by performing a cross-sectional bibliometric analysis using a systematic approach to better understand the publishing trends (including historical vs. emerging themes and research priorities); depth, structure, and evidence-basis of content; and venues for publication. The analysis documented a dramatic increase in biomedical trainee-related publications over time and showed that this area of research is maturing into its own independent field. Results demonstrated that the most frequently published article types in this field are shorter editorial and opinion pieces, and that evidence-based articles are less numerous. However, if current trends continue, projections indicate that by the year 2035, evidence-based articles will be the dominating article type published in this field. Most frequently published topics included career outcomes and workforce characterization and professional development. In recent years, the most cited articles were publications focused on diversity, equity, and inclusion, career outcomes and workforce characterization, and wellness. This study also shows that although a small subset of journals publishes most of this literature, publications are distributed diffusely across a wide range of journals and that surprisingly 68% of these journals have published only a single article on the topic. Further, we noted that the assignment of author- and index-supplied keywords was variable and inconsistent and speculate that this could create challenges to conducting comprehensive literature searches. Recommendations to address this include establishing standard keyword assignment criteria and proposing new index-supplied keywords to improve accessibility of research findings. These changes will be important for bringing visibility of this literature to our community, institutional leaders, national trainee organizations, and funding agencies.

The gastric caecum of larval Aedes aegypti: stimulation of epithelial ion transport by 5-hydroxytryptamine and cAMP.

We report measurements of ion transport across the gastric caecum of larvae of Aedes aegypti, a vector of yellow fever that inhabits a variety of aquatic habitats ranging from freshwater to brackish water. We provide the first measurements of the effect of 5-hydroxytryptamine (5-HT) on transepithelial potential (TEP), luminal ion concentrations and electrochemical potentials, as well as basolateral membrane potential and H+, Na+ and K+ fluxes. TEP, basolateral membrane potential, and H+, K+ and Na+ fluxes across the gastric caeca declined within 3-6 min after isolation of the entire midgut from the larva. 5-HT restored both the TEP and active accumulation of H+, K+ and Na+ in the lumen. Additionally, 5-HT restored H+, K+ and Na+ fluxes across the distal caecum of freshwater larvae, and restored H+ fluxes across the distal caecum of brackish water larvae. There was no effect of 5-HT on ion fluxes across the proximal caecum. We have also shown that 5-HT restores the basolateral membrane potential in cells of the distal, but not proximal, caecum. Effects of 5-HT on TEP and basolateral membrane potential were mimicked by application of cAMP but not by a phorbol ester. We provide a working model which proposes that 5-HT and cAMP stimulate the vacuolar H+-ATPase of the distal caecum. Our results provide evidence that the gastric caecum is functionally distinct from the adjacent anterior midgut and we discuss possible roles of the gastric caecum in osmoregulation. We also describe similarities in the arrangement of ion transporters in the caecum with those of the Malpighian tubules.

Effects of rearing salinity on expression and function of ion-motive ATPases and ion transport across the gastric caecum of Aedes aegypti larvae.

Larvae of Aedes aegypti, the yellow fever vector, inhabit a variety of aquatic habitats ranging from freshwater to brackish water. This study focuses on the gastric caecum of the larvae, an organ that has not been widely studied. We provide the first measurements of H+, K+ and Na+ fluxes at the distal and proximal gastric caecum, and have shown that they differ in the two regions, consistent with previously reported regionalization of ion transporters. Moreover, we have shown that the regionalization of vacuolar H+-ATPase and Na+/K+-ATPase is altered when larvae are reared in brackish water (30% seawater) relative to freshwater. Measurements of luminal Na+ and K+ concentrations also show a 5-fold increase in Na+/K+ ratio in the caecal lumen in larvae reared in brackish water relative to freshwater, whereas transepithelial potential and luminal pH were unchanged. Calculated electrochemical potentials reveal changes in the active accumulation of Na+ and K+ in the lumen of the gastric caecum of freshwater versus brackish water larvae. Together with the results of previous studies of the larval midgut, our results show that the caecum is functionally distinct from the adjacent anterior midgut, and may play an important role in osmoregulation as well as uptake of nutrients.

The roles of V-type H+-ATPase and Na+/K+-ATPase in energizing K+ and H+ transport in larval Drosophila gut epithelia.

We analyzed V-type H+-ATPase (VA) and Na+/K+-ATPase (NKA) along the caeca and midgut of third instar Drosophila larvae using immunohistochemistry and ATPase activity assays. Corresponding H+ and K+ fluxes were characterized using the Scanning Ion-Selective Electrode Technique (SIET), and the roles of transport ATPases in energizing ion transport across the larval gut were investigated by basal application of bafilomycin, a VA inhibitor, and ouabain, a NKA inhibitor. Addition of bafilomycin led to a decrease in H+ absorption along the caeca and midgut except at the copper cells and large flat cell zone of the middle midgut. H+ absorption was decreased by acetazolamide, consistent with carbonic anhydrase activity in all regions except at the large flat cell zone of the middle midgut. Bafilomycin or acetazolamide also led to decreased K+ absorption across the caeca and the anterior midgut. Our data show the dependence of K+ transport on H+ gradients established by the VA in the latter regions, consistent with the presence of a Cation-Proton Antiporter (CPA2) identified in other insect epithelia. Addition of ouabain led to the increase of K+ absorption along the anterior midgut and the large flat cell zone of the middle midgut, suggesting a role for the NKA in these regions. This study shows the importance of both ATPases in driving ion transport across the gut of larval Drosophila.

Electrolyte transport pathways induced in the midgut epithelium of Drosophila melanogaster larvae by commensal gut microbiota and pathogens.

KEY POINTS: The digestive tract of larval and adult Drosophila is an excellent analogue of the mammalian gut. Enterocytes of the posterior midgut are separated by septa, with no paracellular path, and therefore perform both immune and transport functions. Using microperfusion electrophysiology, we show that larvae emerging from the embryo into sterile medium have symmetrical apical and basal membrane conductances while larvae emerging into non-sterile medium have apical membranes fivefold more conductive than basal membranes. The channels inserted into the apical membranes could originate in microbiata or host and mediate recognition of microbes. Entomopathogenic cyclic peptide toxins deplete intracellular ions reversibly, forming transient ion channels that do not conduct water, unlike an ionophore like nystatin that depletes ions irreversibly. We show the feasibility of studying the interaction of a single microbial species, or tractable combinatorials of microbial species, with only enterocytes in the primary epithelial barrier. ABSTRACT: Microbiota colonizing exposed epithelial surfaces are vital for sustenance of metazoan life, but communication between microbiota, epithelial cells and the host immune system is only beginning to be understood. We address this issue in the posterior midgut epithelium of Drosophila larvae where nutrient transport and immune functions are exclusively transcellular. We showed that larvae emerging into a sterile post-embryonic environment have symmetrical apical and basal membranes. In contrast, larvae emerging into non-sterile media, the source of microbiota, have markedly asymmetrical membranes, with apical membrane conductance more than fivefold higher than the basal membrane. As an example of pathogen action, we showed that the entomopathogenic fungal toxin destruxin A (Dx) depleted intracellular ions. Reversibility of action of Dx was verified by bilayer reconstitution in forming transient non-specific channels that conduct ions but not water. Dx was also less effective from the apical side as compared to the basal side of the epithelium. We also showed that intercellular septa are not conductive in non-sterile cells, even though most cells are isopotential. Luminal microbiota therefore impart asymmetry to the epithelium, by activation of apical membrane conductance, enhancing inter-enterocyte communication, separated by insulating septa, via the gut lumen. These results also open the possibility of studying the basis of bidirectional molecular conversation specifically between enterocytes and microbiota that enables discrimination between commensals and pathogens, establishment of the former, and elimination of the latter.