Hypoxia-inducible factor 1 is required for remote ischemic preconditioning of the heart

Abstract

Both preclinical and clinical studies suggest that brief cycles of ischemia and reperfusion in the arm or leg may protect the heart against injury following prolonged coronary artery occlusion and reperfusion, a phenomenon known as remote ischemic preconditioning. Recent studies in mice indicate that increased plasma interleukin-10 (IL-10) levels play an important role in remote ischemic preconditioning induced by clamping the femoral artery for 5 min followed by 5 min of reperfusion for a total of three cycles. In this study, we demonstrate that remote ischemic preconditioning increases plasma IL-10 levels and decreases myocardial infarct size in wild-type mice but not in littermates that are heterozygous for a knockout allele at the locus encoding hypoxia-inducible factor (HIF) 1α. Injection of a recombinant adenovirus encoding a constitutively active form of HIF-1α into mouse hind limb muscle was sufficient to increase plasma IL-10 levels and decrease myocardial infarct size. Exposure of C2C12 mouse myocytes to cyclic hypoxia and reoxygenation rapidly increased levels of IL-10 mRNA, which was blocked by administration of the HIF-1 inhibitor acriflavine or by expression of short hairpin RNA targeting HIF-1α or HIF-1β. Chromatin immunoprecipitation assays demonstrated that binding of HIF-1 to the Il10 gene was induced when myocytes were subjected to cyclic hypoxia and reoxygenation. Taken together, these data indicate that HIF-1 activates Il10 gene transcription and is required for remote ischemic preconditioning.

Keywords: cardiac surgery; cardioprotection; coronary heart disease; myocardial infarction.

Fig. 1.

Analysis of remote IPC (RIPC) of the heart in WT and Hif1a^+/− mice. (A) Schematic of the experimental protocol is shown. WT and Hif1a^+/− (HET) littermate mice were subjected to control sham surgery (CON) or RIPC, which consisted of three 5-min cycles of femoral artery occlusion and reperfusion, followed 24 h later by left coronary artery ligation for 30 min followed by 120 min of reperfusion (I₃₀R₁₂₀). (B) Analysis of MI. The hearts were stained with Evans blue dye, to identify AAR, and triphenyltetrazolium chloride, to determine IS, and then cut into five sections. (C) IS was calculated as a percentage of total LVA or AAR. The AAR as a percentage of LVA is also shown. Data are shown as mean ± SEM (n = 6–10 in each group); **P < 0.01 vs. WT/CON; ^##P < 0.01 vs. WT/RIPC.

Fig. 2.

RIPC induces cardiac AKT phosphorylation and increases plasma levels of IL-10. Mice (n = 4–5 in each group) were subjected to sham surgery (CON) or three 5-min cycles of femoral artery occlusion and reperfusion (RIPC). After 24 h, heart tissue and blood plasma were collected. (A) Heart lysates were prepared and analyzed for levels of phosphorylated (P-AKT) and total (AKT) AKT protein by immunoblot assays (Lower), and the P-AKT:AKT ratio was determined for each sample (mean ± SEM; Upper). *P < 0.05 vs. WT/CON; ^#P < 0.05 vs. WT/RIPC. (B) ELISA for IL-10 was performed on plasma samples (mean ± SEM). **P < 0.01 vs. WT/CON; ^##P < 0.01 vs. WT/RIPC.

Fig. 3.

Effect of HIF-1α gene therapy. WT mice (n = 3 in each group) received an intramuscular injection of a replication-defective recombinant adenovirus encoding E. coli β-galactosidase (AdLacZ) or a constitutively active form of HIF-1α (AdCA5). (A) Twenty-four hours later, blood plasma was collected and ELISA for IL-10 was performed (mean ± SEM). *P < 0.05 vs. AdLacZ. (B) Twenty-four hours later, the mice were subjected to coronary artery occlusion and reperfusion and myocardial IS was determined as a percentage of the total LVA or AAR (mean ± SEM). *P < 0.05; **P < 0.01 vs. AdLacZ.

Fig. 4.

HIF-1 is required for induction of IL-10 mRNA expression in mouse myocytes subjected to CHR. (A) Differentiated C2C12 cells either were subjected to six cycles that each consisted of 5 min at 1% O₂ followed by 5 min at 20% O₂ (CHR) or were exposed to 20% O₂ continuously for 1 h (CON). Whole cell lysates were prepared, and immunoblot assays of HIF-1α and actin were performed. Representative blots from three independent experiments are shown. (B) C2C12 myocytes were exposed to CHR or CON. Reverse transcription−quantitative real-time PCR (RT-qPCR) analyses of IL-10 and Rpl13a mRNA were performed (mean ± SEM, n = 3–4). **P < 0.01 vs. CON. (C) Differentiated C2C12 cells were pretreated with the HIF-1 inhibitor acriflavine or vehicle (0.1% DMSO) for 30 min and exposed to CHR or 20% O₂. RT-qPCR analyses of IL-10 mRNA were performed (mean ± SEM, n = 3). ***P < 0.001 vs. CON/DMSO; ^#P < 0.05 vs. CHR/DMSO. (D and E) C2C12 myocytes were stably transduced with lentivirus expressing a control scrambled shRNA (shSC) or shRNA against HIF-1α (sh1α-2; D) or HIF-1β (sh1β-2; E). C2C12-shSC and C2C12-sh1α-2 (D) or C2C12-sh1β-2 (E) cells were exposed to CHR or CON. Immunoblot assays of HIF-1α or HIF-1β and actin were performed. (F and G) Differentiated C2C12-shSC and C2C12-sh1α-2 (F) or C2C12-sh1β-2 cells (G) were exposed to CHR or CON. RT-qPCR analyses of IL-10, Rpl13a, HIF-1α, or HIF-1β mRNAs were performed (mean ± SEM, n = 4). ***P < 0.001 vs. shSC/CON; ^###P < 0.001 vs. shSC/CHR.

Fig. 5.

HIF-1 binds directly to the Il10 gene in mouse myocytes subjected to CHR. (A) Nucleotide sequence and coordinates of two candidate HIF-1 binding sites located in the 5′-FS and 3′-FS of the mouse Il10 gene. The transcription initiation site (bent arrow) is designated +1. Exons (E) and introns are not drawn to scale. (B–E) Chromatin immunoprecipitation assays. Differentiated C2C12 cells were exposed to CHR or 20% O₂ (CON). Chromatin was immunoprecipitated with total IgG, anti-HIF-1α antibody (B and D), or anti-HIF-1β antibody (C and E) and analyzed by RT-qPCR (mean ± SEM, n = 3–7). *P < 0.05; **P < 0.01 vs. CON.