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The bioactive lipid intermediate palmitoyl CoA (PCoA) can inhibit mitochondrial ADP/ATP transport, though the physiological relevance of this regulation remains unclear. We questioned whether myocardial ischemia provides a pathological setting in which PCoA regulation of ADP/ATP transport would be beneficial, and secondly, whether the chronically elevated lipid content within the diabetic heart could make mitochondria less sensitive to the effects of PCoA. PCoA acutely decreased ADP-stimulated state 3 respiration and increased the apparent Km for ADP twofold. The half maximal inhibitory concentration (IC50) of PCoA in control mitochondria was 22 µM. This inhibitory effect of PCoA on respiration was blunted in diabetic mitochondria, with no significant difference in the Km for ADP in the presence of PCoA, and an increase in the IC50 to 32 µM PCoA. The competitive inhibition by PCoA was localised to the phosphorylation apparatus, particularly the ADP/ATP carrier (AAC). During ischemia, the AAC imports ATP into the mitochondria, where it is hydrolysed by reversal of the ATP synthase, regenerating the membrane potential. Addition of PCoA dose-dependently prevented this wasteful ATP hydrolysis for membrane repolarisation during ischemia, however, this beneficial effect was blunted in diabetic mitochondria. Finally, using 31P-magnetic resonance spectroscopy we demonstrated that diabetic hearts lose ATP more rapidly during ischemia, with a threefold higher ATP decay rate compared with control hearts. In conclusion, PCoA plays a role in protecting mitochondrial energetics during ischemia, by preventing wasteful ATP hydrolysis. However, this beneficial effect is blunted in diabetes, contributing to the impaired energy metabolism seen during myocardial ischemia in the diabetic heart.

Fabry disease is an X-linked lysosomal storage disease caused by α-galactosidase A (α-Gal A) deficiency. Kidney and heart failure are frequent complications in adulthood and greatly contribute to patient morbidity and mortality. Because α-Gal A-deficient mouse models do not recapitulate cardiorenal findings observed in patients, a nonmouse model may be beneficial to our understanding of disease pathogenesis. In this study, we evaluated disease processes in a recently generated Fabry rat model. We found that male Fabry rats weighed significantly less than wild-type (WT) males, whereas female Fabry rats weighed significantly more than WT females. Whereas no difference in female survival was detected, we observed that male Fabry rats had a decreased lifespan. Skin histology revealed that inflammation and lipoatrophy may be chief disease mediators in patients. With respect to the kidney and heart, we found that both organs accumulate α-Gal A substrates, including the established biomarkers, globotriaosylceramide and globotriaosylsphingosine. Longitudinal serum and urine chemistry panels demonstrated pronounced renal tubule dysfunction, which was confirmed histologically. Mitral valve thickening was observed in Fabry rats using echocardiography. We conclude that Fabry rats recapitulate important kidney and heart phenotypes experienced by patients and can be further used to study disease mechanisms and test therapies.-Miller, J. J., Aoki, K., Mascari, C. A., Beltrame, A. K., Sokumbi, O., North, P. E., Tiemeyer, M., Kriegel, A. J., Dahms, N. M., α-Galactosidase A-deficient rats accumulate glycosphingolipids and develop cardiorenal phenotypes of Fabry disease.

Currently, there are no generally applicable noninvasive methods for defining the relationship between atherosclerotic vascular damage and risk of focal thrombosis. Herein, we demonstrate methods to delineate the progression and regression of vascular damage in response to an atherogenic diet by quantifying the in vivo accumulation of semipermeable 200–300 nm perfluorocarbon core nanoparticles (PFC-NP) in ApoE null mouse plaques with [19F] magnetic resonance spectroscopy (MRS). Permeability to PFC-NP remained minimal until 12 weeks on diet, then increased rapidly following 12 weeks, but regressed to baseline within 8 weeks after diet normalization. Markedly accelerated clotting (53.3% decrease in clotting time) was observed in carotid artery preparations of fat-fed mice subjected to photochemical injury as defined by the time to flow cessation. For all mice on and off diet, an inverse linear relationship was observed between the permeability to PFC-NP and accelerated thrombosis (P = 0.02). Translational feasibility for quantifying plaque permeability and vascular damage in vivo was demonstrated with clinical 3 T MRI of PFC-NP accumulating in plaques of atherosclerotic rabbits. These observations suggest that excessive permeability to PFC-NP may indicate prothrombotic risk in damaged atherosclerotic vasculature, which resolves within weeks after dietary therapy.—Palekar, R. U., Jallouk, A. P., Goette, M. J., Chen, J., Myerson, J. W., Allen, J. S., Akk, A., Yang, L., Tu, Y., Miller, M. J., Pham, C. T. N., Wickline, S. A., Pan, H. Quantifying progression and regression of thrombotic risk in experimental atherosclerosis.

Angiotensin (ANGII) and secretin (SCT) share overlapping, interdependent osmoregulatory functions in brain, where SCT peptide/receptor function is required for ANGII action, yet the molecular basis is unknown. Since receptors for these peptides (AT1aR, SCTR) are coexpressed in osmoregulatory centers, a possible mechanism is formation of a cross-class receptor heterocomplex. Here, we demonstrate such a complex and its functional importance to modulate signaling. Association of AT1aR with SCTR reduced ability of SCT to stimulate cyclic adenosine monophosphate (cAMP), with signaling augmented in presence of ANGII or constitutively active AT1aR. Several transmembrane (TM) peptides of these receptors were able to affect their conformation within complexes, reducing receptor BRET signals. AT1aR TM1 affected only formation and activity of the heterocomplex, without effect on homomers of either receptor, and reduced SCT-stimulated cAMP responses in cells expressing both receptors. This peptide was active in vivo by injection into mouse lateral ventricle, thereby suppressing water-drinking behavior after hyperosmotic shock, similar to SCTR knockouts. This supports the interpretation that active conformation of AT1aR is a key modulator of cAMP responses induced by SCT stimulation of SCTR. The SCTR/AT1aR complex is physiologically important, providing differential signaling to SCT in settings of hyperosmolality or food intake, modulated by differences in levels of ANGII.—Lee, L. T. O., Ng, S. Y. L., Chu, J. Y. S., Sekar, R., Harikumar, K. G., Miller, L. J., Chow, B. K. C. Transmembrane peptides as unique tools to demonstrate the in vivo action of a cross-class GPCR heterocomplex.

The aim of this study is to investigate whether frequent cooked broccoli intake can modify the composition and metabolism of gut microbiota, specifically if broccoli feeding improves the ability of...

β-Adrenergic receptor (AR) blockers provide substantial clinical benefits, including improving overall survival and left ventricular (LV) function following myocardial infarction (MI), though the mechanisms remain incompletely defined. The transverse-tubule (T-tubule) system of ventricular myocytes is an important determinant of cardiac excitation-contraction function. T-tubule remodeling occurs early during LV failure. We hypothesized that β-AR blockers prevent T-tubule remodeling and thereby provide therapeutic benefits. A murine model of MI was utilized to examine the effect of β-AR blockers on T-tubule remodeling following LV MI. We applied the in situ imaging of T-tubule structure from Langendorff-perfused intact hearts with laser scanning confocal microscopy. We found that MI caused remarkable T-tubule remodeling near the infarction border zone and moderate LV remodeling remote from the MI. Metoprolol and carvedilol administered 6 d after MI for 4 wk each increased the T-tubule integrity at the remote and border zones. At the molecular level, both β-AR blockers restored border and remote zone expression of junctophilin-2 (JP-2), which is involved in T-tubule organization and formation of the T-tubule/sarcoplasmic reticulum junctions. In contrast, β-AR blockers had no significant effects on caveolin-3 expression. In summary, our data show that β-AR antagonists can protect against T-tubule remodeling after MI, suggesting a novel therapeutic mechanism of action for this drug class. Preservation of JP-2 expression may contribute to the beneficial effects of metoprolol and carvedilol on T-tubule remodeling.—Chen, B., Li, Y., Jiang, S., Xie, Y.-P., Guo, A., Kutschke, W., Zimmerman, K., Weiss, R. M., Miller, F. J., Anderson, M. E., Song, L.-S. β-Adrenergic receptor antagonists ameliorate myocyte T-tubule remodeling following myocardial infarction.

Waardenburg syndrome (WS) is an inherited sensorineural deafness condition in humans caused by melanocyte deficiencies in the inner ear and forelock. Mutation of microphthalmia-associated transcription factor (MITF) is known to produce WS type IIA whereas mutations of either endothelin (EDN) or its receptor endothelin receptor B (EDNRB) produce WS type IV. However, a link between MITF haploinsufficiency and EDN signaling has not yet been established. Here we demonstrate mechanistic connections between EDN and MITF and their functional importance in melanocytes. Addition of EDN to cultured human melanocytes stimulated the phosphorylation of MITF in an EDNRB-dependent manner, which was completely abolished by mitogen-activated protein kinase kinase inhibition. The expression of melanocyte-specific MITF mRNA transcripts was markedly augmented after incubation with EDN1 and was followed by increased expression of MITF protein. Up-regulated expression of MITF was found to be mediated via both the mitogen-activated protein kinase-p90 ribosomal S6 kinase-cAMP response element-binding protein (CREB) and cAMP-protein kinase A-CREB pathways. In addition, EDNRB expression itself was seen to be dependent on MITF. The functional importance of these connections is illustrated by the ability of EDN to stimulate expression of melanocytic pigmentation and proliferation markers in an MITF-dependent fashion. Collectively these data provide mechanistic and epistatic links between MITF and EDN/EDNRB, critical melanocytic survival factors and WS genes.

Team-Based Learning (TBL) is a collaborative teaching method in which students utilize course content to solve challenging problems. A modified version of TBL is used at the University of Louisville School of Medicine. Students complete questions on the Individual Readiness Assurance Test (iRAT) then gather in pre-assigned groups to retake the quiz, given time to utilize their learning resources and discuss each of the questions (Team Readiness Assurance Test-tRAT). Following this discussion, students take an Individual Summative Assessment Test (iSAT) with new questions at a similar cognitive level and content focus. While educational gains of TBL have been shown, student evaluations negatively assessed the teaching method with complaints regarding question difficulty and stress levels. Thus, during implementation of TBL in the School of Dentistry, three main changes were made: (1) The contribution of TBL to the overall grade was reduced (2) TBL questions were cognitively aligned with unit exam questions, and (3) Scratch-off, lottery response cards were used to create a fun, game-like environment. This revised TBL format, compared to the original format, resulted in similar student performance during iRAT and tRAT sessions. However, the revised, low-stress format had significantly higher scores on the iSAT (n=119-161, p

There can be a disconnect between the level of content covered in undergraduate coursework and the expectations of professional-level faculty of their incoming students. Some basic science faculty members may assume that students have a good knowledge base in the material and neglect to appropriately review, while others may spend too much class time reviewing basic material. It was hypothesized that the replacement of introductory didactic physiology lectures with interactive online modules in a first-year D.M.D. course could improve student preparedness prior to lectures. These modules would also allow faculty members to analyze incoming student abilities and save valuable face-to-face class time for alternative teaching strategies. Results indicated that the performance levels of incoming students was poor (57% average on a pre-test), and students often under-predicted their abilities (by 13% on average). Faculty expectations varied greatly between the different content areas, and did not appear to cor...

Diagnostic ultrasound imaging (US) is enhanced by the use of circulating microbubble contrast agents (UCAs), but the interactions between US, UCAs, and vascular tissue are not well understood. We h...

Previously we showed that TCERG1 inhibits the growth arrest and transactivation functions of C/EBPα, through a mechanism that requires the relocalization of TCERG1 to sites of C/EBPα occupancy in the nucleus. TCERG1 contains a unique QA repeat domain, for which no function has been assigned, as well as 3 WW domains within the N-terminal half of TCERG1 which has been shown previously to mediate its relocalization. These domains were explored for their roles in mediating the inhibitory and relocalization activities of TCERG1. While mutations of the WW domains were without effect, deletion of the QA repeat domain in TCERG1 prevented its relocalization in the nucleus (assessed by confocal microscopy), and abrogated its ability to repress both the growth arrest and transactivation functions of C/EBPα. Reducing the number of QA repeats from 38 to 20 had no impact on the activities of TCERG1, while further reduction to 10 resulted in a significant loss of relocalization ability and inhibitory activity towards C/...

Myocardin is a transcriptional coactivator of serum response factor (SRF) responsible for vascular smooth muscle cell (SMC) differentiation. Changes in Nox4 NADPH oxidase expression are associated with changes in myocardin and SRF levels and have been implicated in the phenotypic switching of SMCs. However, the mechanisms underlying this molecular switch in response to vascular injury are poorly understood. We examined the role of microRNAs (miR) in the expression of Nox4, SRF and myocardin in human SMCs. Analysis of the 3’UTR of Nox4 revealed a single binding site for miR-25, and two putative miR-9 binding sites in the 5’ region of the 3’UTR. Treatment of human SMCs with a miR-25 or a miR-9 mimic suppressed mRNA expression of Nox4. We created a Nox4 3’UTR luciferase construct and found that the miR-25 mimic decreased relative light units (RLU) by 40% which was prevented with mutation of the single miR-25 site. The miR-9 mimic decreased Nox4 3’UTR RLU by 60% and mutation of the two miR-9 sites had a syner...

Miller CJ, McNear J, Metz MJ. A comparison of traditional and engaging lecture methods in a large, professional-level course. Adv Physiol Educ 37: 347–355, 2013; doi:10.1152/advan.00050.2013.—In engaging lectures, also referred to as broken or interactive lectures, students are given short periods of lecture followed by “breaks” that can consist of 1-min papers, problem sets, brainstorming sessions, or open discussion. While many studies have shown positive effects when engaging lectures are used in undergraduate settings, the literature surrounding use of the learning technique for professional students is inconclusive. The novelty of this study design allowed a direct comparison of engaging physiology lectures versus didactic lecture formats in the same cohort of 120 first-year School of Dentistry DMD students. All students were taught five physiological systems using traditional lecture methods and six physiological systems using engaging lecture methods. The use of engaging lectures led to a statistically significant higher average on unit exams compared with traditional didactic lectures (8.6% higher, P 0.05). Furthermore, students demonstrated an improved long-term retention of information via higher scores on the comprehensive final exam (22.9% higher in engaging lecture sections, P 0.05). Many qualitative improvements were also indicated via student surveys and evaluations, including an increased perceived effectiveness of lectures, decrease in distractions during lecture, and increased confidence with the material. The development of engaging lecture activities requires a significant amount of instructor preparation and limits the time available to provide traditional lectures. However, the positive results of this study suggest the need for a restructuring of the physiology curriculum to incorporate more engaging lectures to improve both the qualitative experiences and performance levels of professional students.

The effects of growth factors have been shown to depend on the position of a cell in the cell cycle. However, the physiological basis for this phenomenon remains unclear. Here we show that the majority of both CEINGE clone3 (cl3) and human embryonic kidney 293 cells, when arrested in a quiescent phase (G(0)), responded to platelet-derived growth factor BB (PDGF-BB) with non-oscillatory Ca(2+) signals. Furthermore, the same type of Ca(2+) response was also observed in CEINGE cl3 cells (and to a lesser extent in HEK 293 cells) blocked at the G(1)/S boundary. In contrast, CEINGE cl3 cells synchronized in early G(1) or released from G(1)/S arrest responded in an oscillatory fashion. This cell cycle-dependent modulation of Ca(2+) signaling was not observed on epidermal growth factor and G-protein-coupled receptor stimulation and was not due to differences in the expression of PDGF receptors (PDGFRs) during the cell cycle. We demonstrate that inhibition of sphingosine-kinase, which converts sphingosine to sphingosine-1-phosphate, caused G(0) as well as G(1)/S synchronized cells to restore the oscillatory Ca(2+) response to PDGF-BB. In addition, we show that the synthesis of sphingosine and sphingosine-1-phosphate is regulated by the cell cycle and may underlie the differences in Ca(2+) signaling after PDGFR stimulation.

The dorsolateral reticular formation of the caudal medulla, or the lateral tegmental field (LTF), has been classified as the brain's “vomiting center”, as well as an important region in regulating sympathetic outflow. We examined the responses of LTF neurons in cats to rotations of the body that activate vestibular receptors, as well as to stimulation of baroreceptors (through mechanical stretch of the carotid sinus) and gastrointestinal receptors (through the intragastric administration of the emetic compound copper sulfate). Approximately half of the LTF neurons exhibited graviceptive responses to vestibular stimulation, similar to primary afferents innervating otolith organs. The other half of the neurons had complex responses, including spatiotemporal convergence behavior, suggesting that they received convergent inputs from a variety of vestibular receptors. Neurons that received gastrointestinal and baroreceptor inputs had similar complex responses to vestibular stimulation; such responses are expected for neurons that contribute to the generation of motion sickness. LTF units with convergent baroreceptor and vestibular inputs may participate in producing the cardiovascular system components of motion sickness, such as the changes in skin blood flow that result in pallor. The administration of copper sulfate often modulated the gain of responses of LTF neurons to vestibular stimulation, particularly for units whose spontaneous firing rate was altered by infusion of drug (median of 459%). The present results raise the prospect that emetic signals from the gastrointestinal tract modify the processing of vestibular inputs by LTF neurons, thereby affecting the probability that vomiting will occur as a consequence of motion sickness.

Collateral growth (CG) is impaired by aging, but the impairment is reversed by antioxidant treatment. The aim of this study was to determine if NADPH oxidase (Nox) mediated the age-related impairme...