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

Hyperbilirubinemia modulates myocardial function, aortic ejection, and ischemic stress resistance in the Gunn rat

Mildly elevated circulating unconjugated bilirubin (UCB) is associated with protection against hypertension and ischemic heart disease. We assessed whether endogenously elevated bilirubin in Gunn rats modifies cardiovascular function and resistance to ischemic insult. Hearts were assessed ex vivo (Langendorff perfusion) and in vivo (Millar catheterization and echocardiography), and left ventricular myocardial gene expression was measured via quantitative real-time PCR. Ex vivo analysis revealed reduced intrinsic contractility in the Gunn myocardium (+dP/dt: 1,976 ᠶ22 vs. 2,907 ᠳ34 mmHg/s, P < 0.01; -dP/dt: -1,435 ᠳ72 vs. -2,234 ᠴ78 mmHg/s, P < 0.01), which correlated positively with myocardial UCB concentration (P < 0.05). In vivo analyses showed no changes in left ventricular contractile parameters and ejection (fractional shortening and ejection fraction). However, Gunn rats exhibited reductions in the rate of aortic pressure development (3,008 ᠴ61 vs. 4,452 ᠶ44 mmHg/s, P < 0.02), mean aortic velocity (439 ᠶ4 vs. 644 ᠶ2 mm/s, P < 0.01), and aortic volume time integral pressure gradient (2.32 ᠰ.65 vs. 5.72 ᠰ.74 mmHg, P < 0.01), in association with significant aortic dilatation (12-24% increase in aortic diameter, P < 0.05). Ex vivo Gunn hearts exhibited improved ventricular function after 35 min of ischemia and 90 min of reperfusion (63 ᠱ4 vs. 35 ᠱ2%, P < 0.01). These effects were accompanied by increased glutathione peroxidase and reduced superoxide dismutase and phospholamban gene expression in Gunn rat myocardium (P < 0.05). These data collectively indicate that hyperbilirubinemia in Gunn rats 1) reduces intrinsic cardiac contractility, which is compensated for in vivo; 2) induces aortic dilatation, which may beneficially influence aortic ejection velocities and pressures; and 3) may improve myocardial stress resistance in association with beneficial transcriptional changes. These effects may contribute to protection from cardiovascular disease with elevated bilirubin.Griffith Health, School of Medical ScienceNo Full Tex

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

Functional gene networks modified during SLP induction in normoxic myocardium.

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

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

The top 2 cardiovascular-related networks modified by SLP in normoxic myocardium (networks 3 and 4).

<p>Shown are the 3rd and 4th most modified gene networks in SLP hearts. Network 3 is involved in cardiovascular, inflammatory and immune diseases; Network 4 in cardiovascular system development. Transcripts and interactions are coded as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072278#pone-0072278-g003" target="_blank"><b>Figure 3</b></a>.</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