Alternative splicing regulation of membrane trafficking genes during myogenesis.

Alternative splicing transitions occur during organ development, and, in numerous diseases, splicing programs revert to fetal isoform expression. We previously found that extensive splicing changes occur during postnatal mouse heart development in genes encoding proteins involved in vesicle-mediated trafficking. However, the regulatory mechanisms of this splicing-trafficking network are unknown. Here, we found that membrane trafficking genes are alternatively spliced in a tissue-specific manner, with striated muscles exhibiting the highest levels of alternative exon inclusion. Treatment of differentiated muscle cells with chromatin-modifying drugs altered exon inclusion in muscle cells. Examination of several RNA-binding proteins revealed that the poly-pyrimidine tract binding protein 1 (PTBP1) and quaking regulate splicing of trafficking genes during myogenesis, and that removal of PTBP1 motifs prevented PTBP1 from binding its RNA target. These findings enhance our understanding of developmental splicing regulation of membrane trafficking proteins which might have implications for muscle disease pathogenesis.

Deletion of the stress-response protein REDD1 promotes ceramide-induced retinal cell death and JNK activation.

The role of dyslipidemia in the development of retinal dysfunction remains poorly understood. Using an animal model of diet-induced obesity/pre-type 2 diabetes, we investigated molecular defects in the retina arising from consumption of a diet high in saturated fats and sugars ( i.e., a Western diet). We found that feeding mice a Western diet increased the abundance of retinal sphingolipids, attenuated protein kinase B (Akt) phosphorylation, enhanced JNK activation, and increased retinal cell death. When we used palmitate or C6-ceramide (Cer) to assess sphingolipid-mediated signaling in cultured murine and human cells, we observed similar effects on Akt, JNK, and cell death. Furthermore, both Western diet and C6-Cer exposure enhanced expression of the stress-response protein regulated in development and DNA damage response 1 (REDD1) and loss of REDD1 increased C6-Cer-induced JNK activation and cell death. Exogenous REDD1 expression repressed JNK-mediated phosphorylation in cultured cells. We found that thioredoxin-interacting protein (TXNIP) expression was elevated in REDD1-deficient cell lines and C6-Cer promoted TXNIP expression in both wild-type and REDD1-deficient cells. Likewise, TXNIP knockdown attenuated JNK activation and caspase 3 cleavage after either C6-Cer exposure or REDD1 deletion. The results support a model wherein Cer-induced REDD1 expression attenuates TXNIP-dependent JNK activation and retinal cell death.-Dai, W., Miller, W. P., Toro, A. L., Black, A. J., Dierschke, S. K., Feehan, R. P., Kimball, S. R., Dennis, M. D. Deletion of the stress-response protein REDD1 promotes ceramide-induced retinal cell death and JNK activation.

Dietary Fat Quantity and Type Induce Transcriptome-Wide Effects on Alternative Splicing of Pre-mRNA in Rat Skeletal Muscle.

Background: Fat-enriched diets produce metabolic changes in skeletal muscle, which in turn can mediate changes in gene regulation.Objective: We examined the high-fat-diet-induced changes in skeletal muscle gene expression by characterizing variations in pre-mRNA alternative splicing.Methods: Affymetrix Exon Array analysis was performed on the transcriptome of the gastrocnemius/plantaris complex of male obesity-prone Sprague-Dawley rats fed a 10% or 60% fat (lard) diet for 2 or 8 wk. The validation of exon array results was focused on troponin T (Tnnt3). Tnnt3 splice form analyses were extended in studies of rats fed 10% or 30% fat diets across 1- to 8-wk treatment periods and rats fed 10% or 45% fat diets with fat sources from lard or mono- or polyunsaturated fats for 2 wk. Nuclear magnetic resonance (NMR) was used to measure body composition.Results: Consumption of a 60% fat diet for 2 or 8 wk resulted in alternative splicing of 668 and 726 pre-mRNAs, respectively, compared with rats fed a 10% fat diet. Tnnt3 transcripts were alternatively spliced in rats fed a 60% fat diet for either 2 or 8 wk. The high-fat-diet-induced changes in Tnnt3 alternative splicing were observed in rats fed a 30% fat diet across 1- to 8-wk treatment periods. Moreover, this effect depended on fat type, because Tnnt3 alternative splicing occurred in response to 45% fat diets enriched with lard but not in response to diets enriched with mono- or polyunsaturated fatty acids. Fat mass (a proxy for obesity as measured by NMR) did not differ between groups in any study.Conclusions: Rat skeletal muscle responds to overconsumption of dietary fat by modifying gene expression through pre-mRNA alternative splicing. Variations in Tnnt3 alternative splicing occur independently of obesity and are dependent on dietary fat quantity and suggest a role for saturated fatty acids in the high-fat-diet-induced modifications in Tnnt3 alternative splicing.

Fish Oil Enhances T Cell Function and Tumor Infiltration and Is Correlated With a Cancer Prevention Effect in HER-2/neu But Not PyMT Transgenic Mice.

Few studies have explored the effects of omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on immune modulation in murine models of mammary carcinogenesis. HER-2/neu and PyMT mice were randomized to 2 dietary interventions: AIN-93G-based diet with 1) 11% of diet (per gram weight) as corn oil (CO) or 2) 10% of diet as menhaden fish oil plus 1% of diet as corn oil (FO). FO significantly reduced the incidence and multiplicity of tumors (P < 0.001) in HER-2/neu, but not PyMT mice. FO-fed mice had significantly larger splenocyte counts than CO-fed mice in both the HER-2/neu and PyMT models; and in both models this was comprised of an increase in most cell types, including Gr-1(+)/CD11b(+) cells. T cells from FO-fed HER-2/neu mice produced significantly more interleukin-2 (P = 0.004) and interferon-γ (P = 0.012) in response to in vitro stimulation with anti-CD3 (0.5 µg/ml). Lastly, FO-fed HER-2/neu mice had significantly more tumor immune infiltrates than CO-fed mice, including NK1.1(+), F4/80(+), and Gr-1(+)/CD11b(+) cells (P ≤ 0.05). Greater Th1 cytokine production and significantly more tumor immune infiltrates in FO-fed Her2/neu mice may account for the cancer prevention effect of fish oil in this model.

Polarization-based balanced detection for spectral-domain optical coherence tomography.

We present a new design for spectral-domain optical coherence tomography that allows balanced detection using a single camera. The design uses polarization optics to encode the light in reference and sample arms. Two parallel and highly aligned spectra, which carry out-of-phase interference signals, in-phase common noise, and auto-interference terms, are focused on the camera, which performs the digital balanced detection for each wavelength. The optical system is characterized and tested for tissue imaging. Results demonstrate consistent signal gains in depth and suppression of DC and sample auto-interference. The design could be further amended for polarization-sensitive imaging and might demonstrate a market for manufacturing dual-line cameras with analog-balanced detection capability.

Spectral-domain low-coherence interferometry for phase-sensitive measurement of Faraday rotation at multiple depths.

We describe a method for differential phase measurement of Faraday rotation from multiple depth locations simultaneously. A polarization-maintaining fiber-based spectral-domain interferometer that utilizes a low-coherent light source and a single camera is developed. Light decorrelated by the orthogonal channels of the fiber is launched on a sample as two oppositely polarized circular states. These states reflect from sample surfaces and interfere with the corresponding states of the reference arm. A custom spectrometer, which is designed to simplify camera alignment, separates the orthogonal channels and records the interference-related oscillations on both spectra. Inverse Fourier transform of the spectral oscillations in k-space yields complex depth profiles, whose amplitudes and phase difference are related to reflectivity and Faraday rotation within the sample, respectively. Information along a full depth profile is produced at the camera speed without performing an axial scan for a multisurface sample. System sensitivity for the Faraday rotation measurement is 0.86 min of arc. Verdet constants of clear liquids and turbid media are measured at 687 nm.

Visualizing the complex 3D geometry of the perfusion border zone in isolated rabbit heart.

Myocardial infarction, caused by a major blockage of a coronary artery, creates a border zone (BZ) between perfused and nonperfused tissue, which is believed to be the origin of fatal cardiac arrhythmias. We used a combination of optical clearing and polarization-sensitive optical coherence tomography to visualize a three-dimensional organization of the BZ in isolated rabbit hearts (n=5) at the microscopic level with a high spatial resolution. We found that the BZ has a complex three-dimensional structure with nonperfused areas penetrating into perfused tissue with finger-like projections. These "fingers" may play an important role in the initiation and maintenance of ventricular arrhythmias.

Aerobic exercise-mediated changes in the expression of glucocorticoid responsive genes in skeletal muscle differ across the day.

Glucocorticoids are released in response to acute aerobic exercise. The objective was to define changes in the expression of glucocorticoid target genes in skeletal muscle in response to acute aerobic exercise at different times of day. We identified glucocorticoid target genes altered in skeletal muscle by acute exercise by comparing data sets from rodents subjected to acute aerobic exercise in the light or dark cycles to data sets from C2C12 myotubes treated with glucocorticoids. The role of glucocorticoid receptor signaling and REDD1 protein in mediating gene expression was assessed in exercised mice. Changes to expression of glucocorticoid genes were greater when exercise occurred in the dark cycle. REDD1 was required for the induction of genes induced at both times of day. In all, the time of day at which aerobic exercise is conducted dictates changes to the expression of glucocorticoid target genes in skeletal muscle with REDD1 contributing to those changes.

Reconstructing micrometer-scale fiber pathways in the brain: multi-contrast optical coherence tomography based tractography.

Comprehensive understanding of connective neural pathways in the brain has put great challenges on the current imaging techniques, for which three-dimensional (3D) visualization of fiber tracts with high spatiotemporal resolution is desirable. Here we present optical imaging and tractography of rat brain ex-vivo using multi-contrast optical coherence tomography (MC-OCT), which is capable of simultaneously generating depth-resolved images of reflectivity, phase retardance, optic axis orientation and, for in-vivo studies, blood flow images. Using the birefringence property of myelin sheath, nerve fiber tracts as small as a few tens of micrometers can be resolved and neighboring fiber tracts with different orientations can be distinguished in cross-sectional optical slices, 2D en-face images and 3D volumetric images. Combinational contrast of MC-OCT images enables visualization of the spatial architecture and nerve fiber orientations in the brain with unprecedented detail. The results suggest that optical tractography, by virtue of its direct accessibility to nerve fibers, has the potential to validate diffusion magnetic resonance images and investigate structural connections in normal brain and neurological disorders. In addition, an endoscopic MC-OCT may be useful in neurosurgical interventions to aid in placement of deep brain stimulating electrodes.

RNA processing in skeletal muscle biology and disease.

RNA processing encompasses the capping, cleavage, polyadenylation and alternative splicing of pre-mRNA. Proper muscle development relies on precise RNA processing, driven by the coordination between RNA-binding proteins. Recently, skeletal muscle biology has been intensely investigated in terms of RNA processing. High throughput studies paired with deletion of RNA-binding proteins have provided a high-level understanding of the molecular mechanisms controlling the regulation of RNA-processing in skeletal muscle. Furthermore, misregulation of RNA processing is implicated in muscle diseases. In this review, we comprehensively summarize recent studies in skeletal muscle that demonstrated: (i) the importance of RNA processing, (ii) the RNA-binding proteins that are involved, and (iii) diseases associated with defects in RNA processing.

Palmitate- and C6 ceramide-induced Tnnt3 pre-mRNA alternative splicing occurs in a PP2A dependent manner.

BACKGROUND: In a previous study, we showed that consumption of diets enriched in saturated fatty acids causes changes in alternative splicing of pre-mRNAs encoding a number of proteins in rat skeletal muscle, including the one encoding skeletal muscle Troponin T (Tnnt3). However, whether saturated fatty acids act directly on muscle cells to modulate alternative pre-mRNA splicing was not assessed. Moreover, the signaling pathway through which saturated fatty acids act to promote changes in alternative splicing is unknown. Therefore, the objective of the present study was to characterize the signaling pathway through which saturated fatty acids act to modulate Tnnt3 alternative splicing. METHODS: The effects of treatment of L6 myotubes with saturated (palmitate), mono- (oleate), or polyunsaturated (linoleate) fatty acids on alternative splicing of pre-mRNA was assessed using Tnnt3 as a marker gene. RESULTS: Palmitate treatment caused a two-fold change (p < 0.05) in L6 myotube Tnnt3 alternative splicing whereas treatment with either oleate or linoleate had minimal effects compared to control myotubes. Treatment with a downstream metabolite of palmitate, ceramide, had effects similar to palmitate on Tnnt3 alternative splicing and inhibition of de novo ceramide biosynthesis blocked the palmitate-induced alternative splicing changes. The effects of palmitate and ceramide on Tnnt3 alternative splicing were accompanied by a 40-50% reduction in phosphorylation of Akt on S473. However, inhibition of de novo ceramide biosynthesis did not prevent palmitate-induced Akt dephosphorylation, suggesting that palmitate may act in an Akt-independent manner to modulate Tnnt3 alternative splicing. Instead, pre-treatment with okadaic acid at concentrations that selectively inhibit protein phosphatase 2A (PP2A) blocked both palmitate- and ceramide-induced changes in Tnnt3 alternative splicing, suggesting that palmitate and ceramide act through PP2A to modulate Tnnt3 alternative splicing. CONCLUSIONS: Overall, the data show that fatty acid saturation level and ceramides are important factors modulating alternative pre-mRNA splicing through activation of PP2A.

Quantifying three-dimensional optic axis using polarization-sensitive optical coherence tomography.

The optic axis of birefringent samples indicates the direction of optical anisotropy, which should be described in three-dimensional (3-D) space. We present a method to quantify the complete 3-D optic axis orientation calculated from in-plane optic axis measurements from a polarization-sensitive optical coherence tomography system. The in-plane axis orientations with different illumination angles allow the calculation of the necessary polar angle. The method then provides the information to produce the actual birefringence. The method and results from a biological sample are presented.

Regulation of protein and mRNA expression of the mTORC1 repressor REDD1 in response to leucine and serum.

Expression of the mTORC1 repressor, Regulated in DNA Damage and Development 1 (REDD1), is elevated in skeletal muscle during various catabolic conditions including fasting, hindlimb immobilization, and sepsis. Conversely, REDD1 expression is suppressed by anabolic stimuli such as resistance exercise or nutrient consumption following a fast. Though it is known that nutrient consumption reduces REDD1 expression, it is largely unknown how nutrients and hormones individually contribute to the reduction in REDD1 expression. Therefore, the purpose of the present study was to determine how nutrients and hormones individually regulate REDD1 expression. HeLa cells were deprived of leucine or serum for 10 hours, after which either leucine or serum was reintroduced to cell culture medium for 60 minutes. Re-supplementation of either leucine or serum resulted in a reduction in REDD1 protein levels by 34.8 ± 5.8% and 54.1 ± 3.4%, respectively, compared to the deprived conditions. Re-supplementation of leucine or serum to deprived cells also led to a reduction in REDD1 mRNA content by 49.1% ± 2.7% and 65.0 ± 1.4%, respectively, compared to the deprived conditions. Interestingly, rates of REDD1 protein degradation were unaffected by either leucine or serum re-supplementation, as assessed in cells treated with cycloheximide to block protein synthesis. Likewise, addition of leucine or serum to cells treated with Actinomycin D to inhibit gene transcription failed to alter the rate of REDD1 mRNA degradation. The data indicate that the leucine or serum-induced suppression of REDD1 expression occurs independent of changes in the rate of degradation of either the REDD1 protein or mRNA. Thus, the leucine- or serum-induced suppression likely occurs through alternative mechanism(s) such as reduced REDD1 gene transcription and/or mRNA translation.

Optical coherence tomography for cross-sectional imaging of neural activity.

We report a functional optical coherence tomography cross-sectional scanner to detect neural activity using unmyelinated nerves dissected from squid. The nerves, unstained or stained with a voltage-sensitive dye, were imaged in a nerve chamber. Transient phase changes from backscattered light were detected during action potential propagation. The results show that the scanner can provide high spatiotemporal resolution cross-sectional images of neural activity ([Formula: see text]; [Formula: see text]; [Formula: see text] in [Formula: see text]). The advantage of this method compared to monitoring a single depth profile [Formula: see text] is a dramatic increase in the number of available sites that can be measured in two spatial dimensions [Formula: see text] with lateral scanning; therefore, the study demonstrates that two-dimensional monitoring of small-scale functional activity would also be feasible.

Marker-Free Tracking of Facet Capsule Motion Using Polarization-Sensitive Optical Coherence Tomography.

We proposed and tested a method by which surface strains of biological tissues can be captured without the use of fiducial markers by instead, utilizing the inherent structure of the tissue. We used polarization-sensitive optical coherence tomography (PS OCT) to obtain volumetric data through the thickness and across a partial surface of the lumbar facet capsular ligament during three cases of static bending. Reflectivity and phase retardance were calculated from two polarization channels, and a power spectrum analysis was performed on each a-line to extract the dominant banding frequency (a measure of degree of fiber alignment) through the maximum value of the power spectrum (maximum power). Maximum powers of all a-lines for each case were used to create 2D visualizations, which were subsequently tracked via digital image correlation. In-plane strains were calculated from measured 2D deformations and converted to 3D surface strains by including out-of-plane motion obtained from the PS OCT image. In-plane strains correlated with 3D strains (R(2) ≥ 0.95). Using PS OCT for marker-free motion tracking of biological tissues is a promising new technique because it relies on the structural characteristics of the tissue to monitor displacement instead of external fiducial markers.

New system for digital to analog transformation and reconstruction of 12-lead ECGs.

INTRODUCTION: We describe initial validation of a new system for digital to analog conversion (DAC) and reconstruction of 12-lead ECGs. The system utilizes an open and optimized software format with a commensurately optimized DAC hardware configuration to accurately reproduce, from digital files, the original analog electrocardiographic signals of previously instrumented patients. By doing so, the system also ultimately allows for transmission of data collected on one manufacturer's 12-lead ECG hardware/software into that of any other. MATERIALS AND METHODS: To initially validate the system, we compared original and post-DAC re-digitized 12-lead ECG data files (∼5-minutes long) in two types of validation studies in 10 patients. The first type quantitatively compared the total waveform voltage differences between the original and re-digitized data while the second type qualitatively compared the automated electrocardiographic diagnostic statements generated by the original versus re-digitized data. RESULTS: The grand-averaged difference in root mean squared voltage between the original and re-digitized data was 20.8 µV per channel when re-digitization involved the same manufacturer's analog to digital converter (ADC) as the original digitization, and 28.4 µV per channel when it involved a different manufacturer's ADC. Automated diagnostic statements generated by the original versus reconstructed data did not differ when using the diagnostic algorithm from the same manufacturer on whose device the original data were collected, and differed only slightly for just 1 of 10 patients when using a third-party diagnostic algorithm throughout. CONCLUSION: Original analog 12-lead ECG signals can be reconstructed from digital data files with accuracy sufficient for clinical use. Such reconstructions can readily enable automated second opinions for difficult-to-interpret 12-lead ECGs, either locally or remotely through the use of dedicated or cloud-based servers.