Article 1: Cardioprotection by hypoxia-inducible factor 1 alphatransfection in skeletal muscle is dependent on haemoxygenase activity in mice
Aims The present study investigates whether the cardioprotection achieved by gene delivery of hypoxiainduciblefactor-1a (HIF-1a) depends on the downstream factor haem oxygenase (HMOX)-1.
Methods and results Immortalized cardiomyocytes (HL-1 cells) were transfected with HIF-1a or HMOX-1and injured with hydrogen peroxide (H2O2), and death was evaluated by trypan blue staining. Quadricepsmuscles of mice were treated with DNA for HIF-1a and HMOX-1, or sham-treated and electroporated, and 3days later, hearts were isolated and subjected to global ischaemia and reperfusion. Some HIF-1a- andsham-treated mice received the HMOX blocker zinc deuteroporphyrin 2,4-bis-glycol (ZnBG) (n ¼ 6–8 ineach group). HL-1 cells were stimulated with bilirubin or the carbon monoxide donor CORM-2 beforeinjury with H2O2. HL-1 cells which were transfected with HIF-1a or HMOX-1 had an increased survival toH2O2-induced injury compared with empty vector (n ¼ 10–12 per group; P , 0.01 for both). WhenHMOX-1-luciferase reporter mice were treated with HIF-1a in the quadriceps muscle, increased luciferaseactivity was found locally, but nowhere else. Mice pre-treated with HIF-1a or HMOX-1 had a reduced infarctsize, improved post-ischaemic function, and increased serum bilirubin (P , 0.05). ZnBG inhibited all theseeffects afforded by HIF-1a. Stimulation of HL-1 cells with bilirubin and CORM-2 reduced cell death evokedby H2O2 (P , 0.05 for both, n ¼ 11–15 in each group).Conclusion HIF-1a and HMOX-1 provided protection against H2O2-induced damage in HL-1 cells. Remotegene delivery of HIF-1a afforded cardioprotective effects. These were dependent on HMOX activity, asan HMOX blocker abolished the effects, and they were mimicked by pre-treatment with HMOX-1. Downstreamto HMOX-1, bilirubin as well as carbon monoxide may be organ effectors.
Article2: Gene therapy with hypoxia-inducible factor 1 alpha in skeletal muscle is
Aims: Gene therapy of a peripheral organ to protect the heart is clinically attractive. The transcription factorhypoxia-inducible factor 1 alpha (HIF-1α) transactivates cardioprotective genes. We investigated if remotedelivery of DNA encoding for HIF-1α is protective against myocardial ischemia–reperfusion injury in vivo.Main methods: DNA encoding for human HIF-1α was delivered to quadriceps muscles of mice. One week latermyocardial infarction was induced and four weeks later its size was measured. Echocardiography and in vivopressure–volume analysis was performed. Coronary vascularization was evaluated through plastic casting.HL-1 cells, transfected with either HIF-1α or HMOX-1 or administered bilirubin or the carbon monoxide (CO)donor CORM-2, were subjected to lipopolysacharide (LPS)-induced cell death to compare the efficacy oftreatments.Key findings: After four weeks of reperfusion post infarction, animals pretreated with HIF-1α showed reducedinfarct size and left ventricular remodeling (pb0.05, respectively). Fractional shortening was preserved inmice pretreated with HIF-1α (pb0.05). Invasive hemodynamic parameters indicated preserved leftventricular function after HIF-1α (pb0.05), which also induced coronary vascularization (pb0.05). HIF-1αdownstream target heme oxygenase 1 (HMOX-1) was upregulated in skeletal muscle, while serum bilirubinwas increased. Transfection of HL-1 cells with HIF-1α or HMOX-1 and administration of bilirubin or CORM-2comparably salvaged cells from lipopolysacharide (LPS)-induced cell death (all pb0.05).Significance: HIF-1α gene delivery to skeletal muscle preceding myocardial ischemia reduced infarct sizeand postischemic remodeling accompanied by an improved cardiac function and vascularization. Similar toHIF-1α, HMOX-1, bilirubin and CO were protective against LPS-induced injury. This observation may haveclinical potential.cardioprotective in vivo