Skip to main content
. Author manuscript; available in PMC: 2012 Nov 1.
Published in final edited form as: Nat Med. 2012 Apr 15;18(5):774–782. doi: 10.1038/nm.2728

Figure 5. Hif1a as link between adenosine mediated period 2 signaling and metabolism.

Figure 5

(a) Hearts from Hif1a reporter, Per2 reporter or Per2−/−-Hif1a reporter double mutant mice were analyzed for Hif1a or Per2 protein during a 24 h zeitgeber period (*,# p<0.05 over baseline, n=3). (b,c) Per2−/− or cardiac specific Hif1a−/− mice were subjected to IP (IP; 4 cycles of 5 min ischemia followed by 5 min of reperfusion) and Western blot analysis for Hif1a or Per2 protein from the area at risk was performed, respectively. One representative blot of three is displayed. (d) Isolated adult cardiomyocytes from wild-type or Per2−/− mice were exposed to ambient hypoxia [1%, 4h] and analyzed for Hif1a protein. One representative blot of three is displayed. (e) Hif1a reporter or Per2−/−-Hif1a reporter double mutant mice were exposed to IP (IP; 4 cycles of 5 min ischemia followed by 5 min of reperfusion) and the area at risk was analyzed for luciferase activity indicating Hif1a protein (mean±SD; n=4). (f) Transcriptional regulation of glycolytic enzymes in oxygen-stable HIF1A overexpressing HMEC-1 cells with or without siRNA mediated PER2 knockdown. Cells were treated with vehicle or Adora2b agonist BAY 60-6583 and analyzed for transcript levels of phosphofructokinase-m (PFKM), pyruvate kinase (PK), pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase a (LDHA) (mean±SD; n=3). (g) Hearts from wild-type mice were subjected to IP (IP; 4 cycles of 5 min ischemia followed by 5 min of reperfusion) and protein lysates were isolated for native protein complexes using a Per2 antibody covalently coupled (immobilized) onto an amine-reactive resin. Co-immunoprecipitated protein was analyzed using immunoblot against Hif1a. One representative blot of three is displayed. (h,i) Hearts or isolated myocytes from wildtype or Adora2b−/− were exposed to IP (IP; 4 cycles of 5 min ischemia followed by 5 min of reperfusion) or ambient hypoxia [1%, 4h], respectively, and analyzed for Hif1a protein using immunoblot. One representative blot of three is displayed. (j) Transcript levels of phosphofructokinase-m (Pfkm), pyruvate kinase (Pk), pyruvate dehydrogenase kinase 1 (Pdk1) and lactate dehydrogenase a (Ldha) from wildtype or Adora2b−/− mice after IP (IP; 4 cycles of 5 min ischemia followed by 5 min of reperfusion) treatment (mean±SD; n=3).