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 ± 622 vs. 2,907 ± 334 mmHg/s, P < 0.01; -dP/dt: -1,435 ± 372 vs. -2,234 ± 478 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 ± 461 vs. 4,452 ± 644 mmHg/s, P < 0.02), mean aortic velocity (439 ± 64 vs. 644 ± 62 mm/s, P < 0.01), and aortic volume time integral pressure gradient (2.32 ± 0.65 vs. 5.72 ± 0.74 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 ± 14 vs. 35 ± 12%, 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.

Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection.

Cholesterol-rich caveolar microdomains and associated caveolins influence sarcolemmal ion channel and receptor function and protective stress signaling. However, the importance of membrane cholesterol content to cardiovascular function and myocardial responses to ischemia-reperfusion (I/R) and cardioprotective stimuli are unclear. We assessed the effects of graded cholesterol depletion with methyl-ß-cyclodextrin (MßCD) and lifelong knockout (KO) or overexpression (OE) of caveolin-3 (Cav-3) on cardiac function, I/R tolerance, and opioid receptor (OR)-mediated protection. Langendorff-perfused hearts from young male C57Bl/6 mice were untreated or treated with 0.02-1.0 mM MßCD for 25 min to deplete membrane cholesterol and disrupt caveolae. Hearts were subjected to 25-min ischemia/45-min reperfusion, and the cardioprotective effects of morphine applied either acutely or chronically [sustained ligand-activated preconditioning (SLP)] were assessed. MßCD concentration dependently reduced normoxic contractile function and postischemic outcomes in association with graded (10-30%) reductions in sarcolemmal cholesterol. Cardioprotection with acute morphine was abolished with ≥20 µM MßCD, whereas SLP was more robust and only inhibited with ≥200 µM MßCD. Deletion of Cav-3 also reduced, whereas Cav-3 OE improved, myocardial I/R tolerance. Protection via SLP remained equally effective in Cav-3 KO mice and was additive with innate protection arising with Cav-3 OE. These data reveal the membrane cholesterol dependence of normoxic myocardial and coronary function, I/R tolerance, and OR-mediated cardioprotection in murine hearts (all declining with cholesterol depletion). In contrast, baseline function appears insensitive to Cav-3, whereas cardiac I/R tolerance parallels Cav-3 expression. Novel SLP appears unique, being less sensitive to cholesterol depletion than acute OR protection and arising independently of Cav-3 expression.

Early cardiac aging linked to impaired stress-resistance and transcriptional control of stress response, quality control and mitochondrial pathways.

Phenotypic and transcriptomic evidence of early cardiac aging, and associated mechanisms, were investigated in young to middle-aged male mice (C57Bl/6; ages 8, 16, 32, 48 wks). Left ventricular gene expression (profiled via Illumina MouseWG-6 BeadChips), contractile and coronary function, and stress-resistance were assessed in Langendorff perfused hearts under normoxic conditions and following ischemic insult (20 min global ischemia-45 min reperfusion; I-R). Baseline or normoxic contractile function was unaltered by age, while cardiac and coronary 'reserves' (during ß-adrenoceptor stimulation; 1 µM isoproterenol) declined by 48 wks. Resistance to I-R injury fell from 16 to 32 wks. Age-dependent transcriptional changes In un-stressed hearts were limited to 104 genes (>1.3-fold; 0.05 FDR), supporting: up-regulated innate defenses (glutathione and xenobiotic metabolism, chemotaxis, interleukins) and catecholamine secretion; and down-regulated extracellular matrix (ECM), growth factor and survival (PI3K/Akt) signaling. In stressed (post-ischemic) myocardium, ∼15-times as many genes (1528) were age-dependent, grouped into 6 clusters (>1.3-fold change; 0.05 FDR): most changing from 16 wks (45 % up/44 % down), a further 5 % declining from 32 wks. Major age-dependent Biological Processes in I-R hearts reveal: declining ATP metabolism, oxidative phosphorylation, cardiac contraction and morphogenesis, phospholipid metabolism and calcineurin signaling; increasing proteolysis and negative control of MAPK; and mixed changes in nuclear transport and angiogenic genes. Pathway analysis supports reductions in: autophagy, stress response, ER protein processing, mRNA surveillance and ribosome/translation genes; with later falls in mitochondrial biogenesis, oxidative phosphorylation and proteasome genes in I-R hearts. Summarizing, early cardiac aging is evident from 16 to 32 wks in male mice, characterized by: declining cardiovascular reserve and stress-resistance, transcriptomic evidence of constitutive stress and altered catecholamine and survival/growth signaling in healthy hearts; and declining stress response, quality control, mitochondrial energy metabolism and cardiac modeling processes in stressed hearts. These very early changes, potentially key substrate for advanced aging, may inform approaches to healthy aging and cardioprotection in the adult heart.

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.