RNA Sequencing Reveals a Slow to Fast Muscle Fiber Type Transition after Olanzapine Infusion in Rats

<div><p>Second generation antipsychotics (SGAs), like olanzapine, exhibit acute metabolic side effects leading to metabolic inflexibility, hyperglycemia, adiposity and diabetes. Understanding how SGAs affect the skeletal muscle transcriptome could elucidate approaches for mitigating these side effects. Male Sprague-Dawley rats were infused intravenously with vehicle or olanzapine for 24h using a dose leading to a mild hyperglycemia. RNA-Seq was performed on gastrocnemius muscle, followed by alignment of the data with the Rat Genome Assembly 5.0. Olanzapine altered expression of 1347 out of 26407 genes. Genes encoding skeletal muscle fiber-type specific sarcomeric, ion channel, glycolytic, O_2- and Ca²⁺-handling, TCA cycle, vascularization and lipid oxidation proteins and pathways, along with NADH shuttles and LDH isoforms were affected. Bioinformatics analyses indicate that olanzapine decreased the expression of slower and more oxidative fiber type genes (e.g., type 1), while up regulating those for the most glycolytic and least metabolically flexible, fast twitch fiber type, IIb. Protein turnover genes, necessary to bring about transition, were also up regulated. Potential upstream regulators were also identified. Olanzapine appears to be rapidly affecting the muscle transcriptome to bring about a change to a fast-glycolytic fiber type. Such fiber types are more susceptible than slow muscle to atrophy, and such transitions are observed in chronic metabolic diseases. Thus these effects could contribute to the altered body composition and metabolic disease olanzapine causes. A potential interventional strategy is implicated because aerobic exercise, in contrast to resistance exercise, can oppose such slow to fast fiber transitions.</p></div

Statistically significant effects of olanzapine on the expression of genes involved in oxidative lipid metabolism.

<p>The role of these genes in lipid metabolism was determined from RGD, KEGG, Genecards and/or IPA websites as described in Methods. Results are mean ± SE of FPKM gene expression values or normalized fold changes found to be statistically significant using the DEGSeq R package. An asterisk (*) indicates one of 164 prioritized obesity or T2D candidate genes from a previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123966#pone.0123966.ref049" target="_blank">49</a>]. The corresponding molecular identity and database number is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123966#pone.0123966.s002" target="_blank">S1 Table</a>.</p><p>Statistically significant effects of olanzapine on the expression of genes involved in oxidative lipid metabolism.</p

Simplified schematic summarization of muscle fiber types and the pathways affected by olanzapine infusion in skeletal muscle.

<p>Striated skeletal muscle fibers can be categorized into three or more different types such as (1) slow-twitch oxidative (SO, red), type I, (2) fast-twitch oxidative-glycolytic (FOG, intermediate twitch) and in rats (3) the fastest-twitch glycolytic (FG, white), type IIb. Gastrocnemius, the muscle examined in this paper, normally contains all of these. Fiber types differ in twitch speed and metabolic flexibility. They are frequently categorized into the above types based one or more of the following: myosin heavy chain isoform content or myosin ATPase activity or gene expression. Compared to SO fibers, FG fibers have: (a) fewer mitochondria, reduced vascularity and myoglobin (<i>Mb</i>) for O₂ handling making them whiter in appearance compared to the redder SO fibers; (b) lower expression of genes in FFA, glucose and amino acid oxidation pathways, (c) increased expression of most genes in the glycolysis to lactate pathway; (d) different NADH shuttles; (e) fiber type specific expression of specific sarcomere components, and (f) specific isoforms of calcium and monovalent cation handling or transport proteins. Our data suggest that acute exposure to olanzapine is beginning a process that will eventually cause a fiber type transition from a mixed type to a whiter FG (IIb) type. Whiter muscle has been reported to be more susceptible than other fiber types to atrophy, and such fiber type transitions changes are associated with metabolic disease and obesity.</p

Comparison of RNA-Seq to QT-RTPCR for selected gastrocnemius muscle genes affected by olanzapine infusion.

<p>Genes significantly affected by olanzapine infusion based on RNA-Seq were selected for QT-RTPCR analysis using TaqMan gene expression assays using the same preparation of RNA. Genes were selected so that different FKPM size bins (1–10: <i>OSTN</i>; 10–99: <i>OSTN</i>, <i>Casq2</i>, <i>Lpl</i>; 100–999: <i>Lpl</i>, <i>Atp2a2</i>, <i>Tnni1</i>, 1000–9000: Tnni, Pvalb; >9000: Tpm1) were represented. The bars show mean± SE. Statistical significance was determined using the DEGSeq R package, which takes into consideration the variability of all of the genes analyzed. QT-RTPCR differences were analyzed using Student’s t-test. Asterisk symbols indicate a significant difference compared to control, *: p <0.05, **: p<0.01, ***: p<0.001)</p

Rod TSS are conserved between human and mouse.

<p><b>A, B, C.</b> Combined genome-wide tracks of chromatin features in mouse retina (developmental changes in H3K4me2 occupancy (PN1, PN15 and RD1 PN30), CRX-binding (PN56), NRL-binding (PN28), developmental changes in DHS (1D, 1W, 8W)) and RNA-seq for human retina for genes TNFAIP3 (<b>A</b>), PLA2G5 (<b>B</b>) and CACNB2 (<b>C</b>) genes. Predicted rod TSS that shows conservation between mouse and human depicted as a black box.</p

Olanzapine shifts expression of genes associated with skeletal muscle fiber type.

<p>Isoforms of fast and slow twitch muscle fiber genes with statistically significant changes when comparing Vehicle and Olanzapine groups are indicated by the presence of a normalized fold change value. Data are means plus or minus (±) SE of the fragments per kilobase of exon per million fragments mapped values (FKPM). N.S. indicates no significant difference. An asterisk(*) indicates a prioritized T2D or obesity candidate gene [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123966#pone.0123966.ref049" target="_blank">49</a>].</p><p>Olanzapine shifts expression of genes associated with skeletal muscle fiber type.</p

Canonical EIF2-signaling pathway/ protein synthetic machinery genes statistically altered by olanzapine infusion in gastrocnemius.

<p>Genes from the canonical pathway from Ingenuity Pathway Analysis that were significantly affected by olanzapine treatment are shown.</p><p>Canonical EIF2-signaling pathway/ protein synthetic machinery genes statistically altered by olanzapine infusion in gastrocnemius.</p

Identification and prediction of alternative transcription start sites that generate rod photoreceptor-specific transcripts from ubiquitously expressed genes

<div><p>Transcriptome complexity is substantially increased by the use of multiple transcription start sites for a given gene. By utilizing a rod photoreceptor-specific chromatin signature, and the RefSeq database of established transcription start sites, we have identified essentially all known rod photoreceptor genes as well as a group of novel genes that have a high probability of being expressed in rod photoreceptors. Approximately half of these novel rod genes are transcribed into multiple mRNA and/or protein isoforms through alternative transcriptional start sites (ATSS), only one of which has a rod-specific epigenetic signature and gives rise to a rod transcript. This suggests that, during retina development, some genes use ATSS to regulate cell type and temporal specificity, effectively generating a rod transcript from otherwise ubiquitously expressed genes. Biological confirmation of the relationship between epigenetic signatures and gene expression, as well as comparison of our genome-wide chromatin signature maps with available data sets for retina, namely a ChIP-on-Chip study of Polymerase-II (Pol-II) binding sites, ChIP-Seq studies for NRL- and CRX- binding sites and DHS (University of Washington data, available on UCSC mouse Genome Browser as a part of ENCODE project) fully support our hypothesis and together accurately identify and predict an array of new rod transcripts. The same approach was used to identify a number of TSS that are not currently in RefSeq. Biological conformation of the use of some of these TSS suggests that this method will be valuable for exploring the range of transcriptional complexity in many tissues. Comparison of mouse and human genome-wide data indicates that most of these alternate TSS appear to be present in both species, indicating that our approach can be useful for identification of regulatory regions that might play a role in human retinal disease.</p></div

Association of rod photoreceptor transcription factors CRX and NRL with rod TSS.

<p><b>A, C.</b> Number of TSS with different CRX (<b>A</b>) and NRL (<b>C</b>) binding for rod-specific genes with single TSS (upper panel) and for rod (middle panel) or common (bottom panel) TSS of retina genes with multiple TSS. Blue–no TF-binding; red–TF-binding. <b>B, D.</b> CRX (<b>B</b>) and NRL (<b>D</b>) accumulation at TSS (normalized number of reads at TSS+/-1000bp) for rod and common TSS of retina genes with multiple TSS, compared with control groups of genes (non-rod, cell-cycle and synapse). ***—p < 0.0001. <b>E, G.</b> Combined genome-wide tracks of chromatin features, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#pone.0179230.g001" target="_blank">Fig <b>1B</b></a> for <i>Wdr17</i> (<b>E</b>) and <i>Tnfaip3</i> (<b>G</b>) genes. Common TSS is depicted as C/ red box, rod TSS–as R/ blue box. Position of the specific primer sets and PCR product that were used to assess and distinguish TSS-specific gene expression by RT-PCR depicted as double arrow below gene maps. <b>F, H.</b> Relative gene expressions from rod and common TSS by RT-PCR with primer pairs depicted at <b>E and G</b> for <i>Wdr17</i> (<b>F</b>) and <i>Tnfaip3</i> (<b>H</b>) for mouse retina samples at PN1, PN15, adult and RD1 mutant, compare with mouse liver. For comparison, normalized to <i>Gapdh</i> delta Ct values for each sample are in table below. Experiments done in duplicates with three technical replicas; ***—p < 0.0001.</p

Epigenetic signature predicts employment of ATSS of ubiquitous gene in tissue-specific manner.

<p><b>A.</b> Pie chart presentation for number of TSS for different groups of rod-specific genes. <b>B.</b> All TSS for rod-specific genes were clustered based on chromatin signature around TSS+/-1000bp for following features: developmental changes in H3K4me2 occupancy (E17, PN1, PN7, PN15 and RD1 PN30), CRX-binding (PN56), NRL-binding (PN28), developmental changes in DHS (1D, 1W, 8W), developmental changes in PolII (PN2, PN25) as clustering criteria (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179230#sec002" target="_blank">methods</a> for details). <b>C, D.</b> Combined genome-wide tracks of chromatin features, as in <b>B</b> for <i>Gnb5</i> and <i>Rorb</i> genes. Common TSS is depicted as C/ red box, rod TSS–as R/ blue box.</p