Unique Transcriptional Profile of Sustained Ligand-Activated Preconditioning in Pre- and Post-Ischemic Myocardium

BACKGROUND: Opioidergic SLP (sustained ligand-activated preconditioning) induced by 3–5 days of opioid receptor (OR) agonism induces persistent protection against ischemia-reperfusion (I-R) injury in young and aged hearts, and is mechanistically distinct from conventional preconditioning responses. We thus applied unbiased gene-array interrogation to identify molecular effects of SLP in pre- and post-ischemic myocardium. METHODOLOGY/PRINCIPAL FINDINGS: Male C57Bl/6 mice were implanted with 75 mg morphine or placebo pellets for 5 days. Resultant SLP did not modify cardiac function, and markedly reduced dysfunction and injury in perfused hearts subjected to 25 min ischemia/45 min reperfusion. Microarray analysis identified 14 up- and 86 down-regulated genes in normoxic hearts from SLP mice (≥1.3-fold change, FDR≤5%). Induced genes encoded sarcomeric/contractile proteins (Myh7, Mybpc3,Myom2,Des), natriuretic peptides (Nppa,Nppb) and stress-signaling elements (Csda,Ptgds). Highly repressed genes primarily encoded chemokines (Ccl2,Ccl4,Ccl7,Ccl9,Ccl13,Ccl3l3,Cxcl3), cytokines (Il1b,Il6,Tnf) and other proteins involved in inflammation/immunity (C3,Cd74,Cd83, Cd86,Hla-dbq1,Hla-drb1,Saa1,Selp,Serpina3), together with endoplasmic stress proteins (known: Dnajb1,Herpud1,Socs3; putative: Il6, Gadd45g,Rcan1) and transcriptional controllers (Egr2,Egr3, Fos,Hmox1,Nfkbid). Biological themes modified thus related to inflammation/immunity, together with cellular/cardiovascular movement and development. SLP also modified the transcriptional response to I-R (46 genes uniquely altered post-ischemia), which may influence later infarction/remodeling. This included up-regulated determinants of cellular resistance to oxidant (Mgst3,Gstm1,Gstm2) and other forms of stress (Xirp1,Ankrd1,Clu), and repression of stress-response genes (Hspa1a,Hspd1,Hsp90aa,Hsph1,Serpinh1) and Txnip. CONCLUSIONS: Protection via SLP is associated with transcriptional repression of inflammation/immunity, up-regulation of sarcomeric elements and natriuretic peptides, and modulation of cell stress, growth and development, while conventional protective molecules are unaltered

Relationship between transcript and protein expression changes for cardiac MYH7 and ANP.

<p>Data are shown for myocardial: <b>A)</b><i>Myh7</i> and MYH7 transcript and protein levels, respectively; and <b>B)</b><i>Nppa</i> and ANP transcript and protein levels, respectively (<i>n</i> = 6 per group). <i>ND</i>; not detected (MYH7 was un-detectable in the placebo group; ANP was un-detectable in the cytosolic fraction). Values are mean±S.E.M. *, P<0.05 <i>vs.</i> Placebo.</p

Functional gene networks modified during SLP induction in normoxic myocardium.

<p>Functional gene networks modified during SLP induction in normoxic myocardium.</p

Cardioprotective effects of OR-dependent SLP.

<p>Data are shown for contractile recoveries and cell death following 25 min ischemia and 45 min reperfusion in isolated hearts from placebo <i>vs</i>. SLP treated mice (<i>n</i> = 8 per group). Shown are recoveries of left ventricular developed pressure (% of baseline) and left ventricular end-diastolic pressure (mmHg), together with total post-ischemic washout of cellular LDH. Values are mean±S.E.M. *, P<0.05 <i>vs.</i> Placebo.</p

Baseline function in Langendorff hearts from SLP and placebo mice.

<p>Data were acquired after 30 min aerobic perfusion (at a fixed heart rate of 420 bpm). Data are means±S.E.M. There were no significant differences in baseline (pre-ischemic) functional measures between groups. LVEDP, left ventricular end-diastolic pressure; LVDP, left ventricular developed pressure; dP/dt, differential of ventricular pressure development or relaxation over time.</p

The top functional gene groupings sensitive to SLP induction in normoxic myocardium.

<p>Functional groupings of transcripts differentially modified by SLP in normoxic tissue (also shown are P-values, and numbers of involved genes). Groupings from IPA analysis are categorized into molecular and cellular functions, physiological system development and function, and disease and disorder (complete functional gene grouping data can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072278#pone.0072278.s003" target="_blank">Table S3</a></b>).</p

Functional gene networks modified by SLP in post-ischemic myocardium.

<p>Functional gene networks modified by SLP in post-ischemic myocardium.</p

Validation of microarray assay data by RT-qPCR.

<p>Shown are expression changes determined via RT-qPCR and microarray analysis for: <b>A)</b> SLP <i>vs</i> placebo responses in normoxic myocardium; and <b>B)</b> SLP <i>vs.</i> placebo responses in post-ischemic myocardium. Data are expressed as means ± S.E.M. (<i>n</i> = 6 per group). Linear regression analysis of these data yielded a significant and strongly positive correlation (r² = 0.95): RT-qPCR expression = (1.351×microarray expression) - 0.047 (the slope factor >1 indicative of a predictably greater dynamic range for RT-qPCR analysis).</p

The top 2 networks modified by SLP in normoxic myocardium (networks 1 and 2, both involved in immunity/inflammation).

<p>Shown are the 2 most modified gene networks in SLP hearts. Network 1 is involved in hematological development and cellular movement/immune cell trafficking; Network 2 in antigen presentation and immune/inflammatory function. Transcripts are color-coded according to expression changes (green, up-regulated; red, down-regulated). Grey highlights molecules present in the dataset (FDR≤5%) that did not meet the ≥1.3-fold cut-off criteria. White indicates predicted molecules computationally incorporated into networks based on evidence within the IPA knowledge base. Lines between molecules indicate direct molecular connections.</p

The top functional gene groupings sensitive to SLP in post-ischemic myocardium.

<p>Functional groupings of transcripts differentially modified by SLP in post-ischemic tissue (also shown are P-values, and numbers of involved genes). Groupings from IPA analysis are categorized into molecular and cellular functions, physiological system development and function, and disease and disorders (complete functional gene grouping data can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072278#pone.0072278.s004" target="_blank">Table S4</a></b>).</p