Hypoxia-inducible factor 2-alpha-dependent induction of amphiregulin dampens myocardial ischemia-reperfusion injury

Fig. 1. Contribution of myocyte-specific hypoxia-inducible factor (HIF) isoforms Hif1a or Hif2a to cardioprotection. a Schematic of breeding approach to generate mice with induced myocyte-specific HIF deletions used in subsequent studies. Hif1aloxP/loxP or Hif2aloxP/loxP mice were crossed with Cre-recombinase expressing mice under the control of Myosin-heavy chain promoter (Myosin-Cre+); these mice express cre-recombinase under the control of a tamoxifen-inducer. Control animals (Myosin-Cre+), Hif1aloxP/loxPMyosin-Cre+ or Hif2aloxP/loxPMyosin-Cre+ received a daily dose of 1 mg i.p. Tamoxifen five consecutive days to induce the Cre-recombinase activity. After 7 days, animals underwent experimental protocol (60 min of in situ myocardial ischemia followed by 120 min of reperfusion). b HIF1-alpha or HIF2-alpha immunoblot analysis from homogenized myocardial tissue, harvested from male Myosin-Cre+ and Hif1aloxP/loxPMyosin-Cre+ or Hif2aloxP/loxPMyosin-Cre+ mice, matched in age and weight. Mice underwent a thoracotomy with no further treatment(-I) or 60 min of myocardial ischemia (+I) followed by 120 min reperfusion; β-Actin (ACTb) served as a loading control. One representative blot out of…

Fig. 2. Hif2a-dependent induction of amphiregulin during myocardial injury. a, b Myosin-Cre+ or Hif2aloxP/loxPMyosin-Cre+ underwent 45 min myocardial ischemia, followed by 120 min reperfusion. After harvest of the area-at-risk, total RNA was probed for transcriptional changes using a microarray technique (Agilent-014868 Whole Mouse Genome Microarray 4x44K G4122F Probe Name version). Transcriptional changes were calculated in relation to the myocardial baseline expression in untreated littermate controls of the same genotype. Array data have been deposited at http://www.ncbi.nlm.nih.gov/geo/ (accession number GSE67308). a List of “top hits” of genes transcriptionally induced in the Area-At-Risk in Myosin-Cre+ but unchanged in Hif2aloxP/loxPMyosin-Cre+ (n = 4 per group, mean ± SD). Note: the most prominent differential regulation was found for the epidermal growth factor amphiregulin (Areg). b Analysis of relevant networks in the IPA (Ingenuity Pathway Analysis®) library most significant to the dataset. Note: selective induction of Areg c Myosin-Cre+, Hif1aloxP/loxPMyosin-Cre+, or Hif2aloxP/loxPMyosin-Cre+ underwent 60 min myocardial ischemia, following 120 min reperfusion. Total RNA from…

Fig. 3. Functional role of HIF2A in the transcriptional regulation of amphiregulin (AREG) in human cardiac myocytes. a–e Human cardiac myocytes (HCM) underwent control, HIF1A-specific or HIF2a-specific short hairpin RNA (shRNA) lentiviral transfection to suppress transcription of hypoxia-inducible factors HIF1A or HIF2A. a, b shRNA-transfected HCM were exposed to ambient hypoxia (1% oxygen) for 16 h and analysis of transcript changes by RT-PCR of HIF1A or HIF2A, respectively. Transcriptional changes were calculated relative to an internal housekeeping gene (Actin-b). Data are expressed as mean fold change ± SD compared to normoxic cells (n = 6 per group). c Immunoblot for HIF1A or HIF2A from shRNA-transfected normoxic or hypoxic HCM. β-Actin (ACTb) served as a loading control. d, e Quantification by densitometry of the HIF1-alpha or HIF2-alpha immunoblot results relative to ACTb. Data are expressed as mean fold change ± SD normalized to shControl compared by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3 per group; d F5,11 = 14.48, p = 0.0002; e F5,11 = 6,726, p = 0.0042). f HCM transfected with shRNA directed against HIF1A, HIF2A or control shRNA were exposed to…

Fig. 4. Cardiac amphiregulin protein levels in myocardial tissues from healthy controls or patients with ischemic heart disease (IHD). a Amphiregulin (AREG) protein levels were determined by immunoblot after total protein isolation from control, healthy myocardial tissue (labeled C), or samples from patients with ischemic heart disease (labeled IHD). β-Actin (ACTb) served as a loading control. b Quantification by densitometry of the AREG immunoblot results relative to ACTb (n = 10 in both groups; data compared by two-sided Mann–Whitney test)

Fig. 5. Myocardial tissue injury in Areg−/− mice exposed to myocardial ischemia and reperfusion injury. a–f Experimental animals were exposed to 60 min of myocardial ischemia, followed by 120 min of reperfusion; infarct sizes were measured by double staining with Evan’s blue and triphenyltetrazolium chloride and serum samples were collected. All infarct sizes are presented as the percentage of infarcted tissue in relation to the area-at-risk. Serum troponin levels were determined by ELISA (a). Infarct sizes were determined in gene-targeted mice for amphiregulin (Areg−/− mice) or isogenic control animals (Areg+/+ mice) (n = 5 per group). b Representative infarct staining from Areg−/− or Areg+/+. c Troponin serum levels in Areg−/− or Areg+/+ mice (n = 5 per group). d Reconstitution of Areg−/− mice with recombinant AREG. Infarct sizes in Areg−/− treated with vehicle or 10 μg of recombinant murine Areg, administered over 15 min period by an indwelling catheter (n = 6 per group). e Representative infarct staining from Areg−/− mice treated with vehicle or 10 µg recombinant murine Areg. f Troponin serum levels after myocardial IR injury of Areg−/− mice treated with vehicle or 10 µg recombinant…

Fig. 6. Pharmacologic inhibition of the amphiregulin receptor ErbB1 during murine myocardial ischemia–reperfusion injury. a–c Mice underwent 60 min of myocardial ischemia, followed by 120 min of reperfusion; infarct sizes were measured by double staining with Evan’s blue and triphenyltetrazolium chloride and serum samples were collected. All infarct sizes are presented as the percentage of infarcted tissue in relation to the area-at-risk. Serum Troponin levels were determined by ELISA. a Infarct sizes of C57/BL6 mice after 60 min of ischemia and 120 min reperfusion, treated prior to myocardial ischemia reperfusion with vehicle or an ERBB1-specific inhibitor (AG1478), administered 15 min prior to ischemia via an indwelling arterial catheter. Data presented as the percentage of infarcted area in relation to area-at-risk (n = 4 per group). b Representative infarct staining of C57/BL6 treated with vehicle or AG1478. c Troponin serum levels of C57/BL6 mice, after 60 min ischemia, 120 min reperfusion, treated with vehicle or AG1478 (n = 5 per group). d Infarct sizes of C57/BL6 mice after 60 min of ischemia and 120 min reperfusion, treated 15 min prior to ischemia via an indwelling arterial…

Fig. 7. Treatment of murine myocardial ischemia and reperfusion injury with recombinant amphiregulin. a–c Wild-type were exposed to 60 min of myocardial ischemia, followed by 120 min of reperfusion; infarct sizes were measured by double staining with Evan’s blue and triphenyltetrazolium chloride and serum samples were collected. All Infarct sizes are presented as the percentage of infarcted tissue in relation to the area-at-risk. Serum troponin levels were determined by ELISA. a Infarct sizes of C57/BL6 mice after 60 min of ischemia and 120 min reperfusion, treated with vehicle or 10 µg recombinant murine AREG, administered over a 15 min period via an indwelling arterial catheter. Data presented are the percentage of infarcted area in relation to area-at-risk. Statistical significance assessed by two-sided, unpaired Student’s t-test (n = 7 per group; data presented as mean ± SD). b Representative infarct staining of C57/BL6 treated with vehicle or 10 µg recombinant murine AREG. c Serum troponin levels of C57/BL6 mice, after 60 min ischemia, 120 min reperfusion, treated with vehicle or 10 µg recombinant murine Areg. Statistical significance assessed by two-sided, unpaired Student’s t-test…

Fig. 8. Reconstitution of cardiomyocyte-specific Hif2a-deficient mice with recombinant amphiregulin. a–c Hif2aloxP/loxPMyosin-Cre+ of similar age, gender, and weight as control mice were exposed to 60 min of myocardial ischemia, followed by 120 min of reperfusion; infarct sizes were measured by double staining with Evan’s blue and triphenyltetrazolium chloride and serum samples were collected. All Infarct sizes are presented as the percentage of infarcted tissue in relation to the area-at-risk. Serum troponin levels were determined by ELISA. Note that data in (a–c) in the “no treatment group” are used in part in Fig. 1c–e to display and analyze IR injury from similar experimental conditions. a Infarct sizes of Hif2aloxP/loxPMyosin-Cre+ mice after 60 min of ischemia and 120 min reperfusion that were pre-treated with 10 µg recombinant murine AREG administered over a 15 min period via an indwelling arterial catheter or received no pharmacologic intervention. Data presented as the percentage of infarcted area in relation to area-at-risk. Statistical significance assessed by two-sided, unpaired Student’s t-test (no treatment n = 6; 10 μg AREG n = 9, data presented as mean ± SD). b…