Heme Oxygenase-1 Induction Improves Cardiac Function following Myocardial Ischemia by Reducing Oxidative Stress

Background Oxidative stress plays a key role in exacerbating diabetes and cardiovascular disease. Heme oxygenase-1 (HO-1), a stress response protein, is cytoprotective, but its role in post myocardial infarction (MI) and diabetes is not fully characterized. We aimed to investigate the protection and the mechanisms of HO-1 induction in cardiomyocytes subjected to hypoxia and in diabetic mice subjected to LAD ligation. Methods In vitro: cultured cardiomyocytes were treated with cobalt-protoporphyrin (CoPP) and tin protoporphyrin (SnPP) prior to hypoxic stress. In vivo: CoPP treated streptozotocin-induced diabetic mice were subjected to LAD ligation for 2/24 h. Cardiac function, histology, biochemical damage markers and signaling pathways were measured. Results HO-1 induction lowered release of lactate dehydrogenase (LDH) and creatine phospho kinase (CK), decreased propidium iodide staining, improved cell morphology and preserved mitochondrial membrane potential in cardiomyocytes. In diabetic mice, Fractional Shortening (FS) was lower than non-diabetic mice (35±1%vs.41±2, respectively p\u3c0.05). CoPP-treated diabetic animals improved cardiac function (43±2% p\u3c0.01), reduced CK, Troponin T levels and infarct size compared to non-treated diabetic mice (P\u3c0.01, P\u3c0.001, P\u3c0.01 respectively). CoPP-enhanced HO-1 protein levels and reduced oxidative stress in diabetic animals, as indicated by the decrease in superoxide levels in cardiac tissues and plasma TNFα levels (p\u3c0.05). The increased levels of HO-1 by CoPP treatment after LAD ligation led to a shift of the Bcl-2/bax ratio towards the antiapoptotic process (p\u3c0.05). CoPP significantly increased the expression levels of pAKT and pGSK3β (p\u3c0.05) in cardiomyocytes and in diabetic mice with MI. SnPP abolished CoPP\u27s cardioprotective effects. Conclusions HO-1 induction plays a role in cardioprotection against hypoxic damage in cardiomyocytes and in reducing post ischemic cardiac damage in the diabetic heart as proved by the increased levels of pAKT with a concomitant inhibition of pGSK3β leading to preserved mitochondrial membrane potential

Heme oxygenase-1 induction improves cardiac function following myocardial ischemia by reducing oxidative stress.

Oxidative stress plays a key role in exacerbating diabetes and cardiovascular disease. Heme oxygenase-1 (HO-1), a stress response protein, is cytoprotective, but its role in post myocardial infarction (MI) and diabetes is not fully characterized. We aimed to investigate the protection and the mechanisms of HO-1 induction in cardiomyocytes subjected to hypoxia and in diabetic mice subjected to LAD ligation.In vitro: cultured cardiomyocytes were treated with cobalt-protoporphyrin (CoPP) and tin protoporphyrin (SnPP) prior to hypoxic stress. In vivo: CoPP treated streptozotocin-induced diabetic mice were subjected to LAD ligation for 2/24 h. Cardiac function, histology, biochemical damage markers and signaling pathways were measured.HO-1 induction lowered release of lactate dehydrogenase (LDH) and creatine phospho kinase (CK), decreased propidium iodide staining, improved cell morphology and preserved mitochondrial membrane potential in cardiomyocytes. In diabetic mice, Fractional Shortening (FS) was lower than non-diabetic mice (35±1%vs.41±2, respectively p<0.05). CoPP-treated diabetic animals improved cardiac function (43±2% p<0.01), reduced CK, Troponin T levels and infarct size compared to non-treated diabetic mice (P<0.01, P<0.001, P<0.01 respectively). CoPP-enhanced HO-1 protein levels and reduced oxidative stress in diabetic animals, as indicated by the decrease in superoxide levels in cardiac tissues and plasma TNFα levels (p<0.05). The increased levels of HO-1 by CoPP treatment after LAD ligation led to a shift of the Bcl-2/bax ratio towards the antiapoptotic process (p<0.05). CoPP significantly increased the expression levels of pAKT and pGSK3β (p<0.05) in cardiomyocytes and in diabetic mice with MI. SnPP abolished CoPP's cardioprotective effects.HO-1 induction plays a role in cardioprotection against hypoxic damage in cardiomyocytes and in reducing post ischemic cardiac damage in the diabetic heart as proved by the increased levels of pAKT with a concomitant inhibition of pGSK3β leading to preserved mitochondrial membrane potential

experimental protocol: Schematic illustration of the protocols used in the in vivo experiments.

<p>experimental protocol: Schematic illustration of the protocols used in the in vivo experiments.</p

CoPP increases Cell viability and preserved mitochondrial membrane potential in cardiomyocytes culture.

<p>A: Morphology of CoPP-pretreated cardiomyocytes Treatment with CoPP preserved an improved morphological appearance of the cardiomyocytes after 2 h of hypoxia compared with the untreated group. SnPP administration abolished the beneficial effect of CoPP-pretreatment. Scale bar, 20 μm. B: The viability of CoPP-treated cardiomyocytes subjected to hypoxia Cultured cardiomyocytes were subjected to 2 h hypoxia with or without CoPP. The cells were stained with propidium iodide (PI), which marks damaged cells. The number of dead cells was lower in the group treated with CoPP compared with the untreated group. SnPP reduced the beneficial effect of CoPP-pretreatment. Data are means of at least 3 replicates in 3 separate experiments. Values are means ± SD (<sup>p<0.01 vs. normoxia #p<0.01 vs. hypoxia, *p<0.01 vs. hyp +CoPP). C: Mitochondrial function of CoPP-treated cardiomyocytes Rat cultured cardiomyocytes were subjected to 2 h hypoxic conditions with or without CoPP. D: The cells were stained with DASPMI. Hypoxic cells treated with CoPP exhibited normal mitochondrial membrane potential. SnPP administration reduced the beneficial effect of CoPP. Data are means of at least three replicates in three separate experiments. Values are means ± SD (<i>p</i><0.01 vs. normoxia #<i>p</i><0.01 vs. hypoxia, *<i>p</i><0.01 vs. hyp +CoPP, magnification *40).</p

Changes in fractional shortening and left ventricle dimensions in Diabetic Mice.

<p>A: LV end-systolic diameters 24 hour post -MI. The LV end-systolic diameter increased in diabetic mice compared with non-diabetic values (*<i>p</i><0 05 vs. non diabetic # <i>p</i><0.05 vs. diabetes values are presented as means ± SD). B: Fractional shortening (FS): CoPP treatment increased FS compared to the untreated diabetic ones SnPP abolished the beneficial effect (*<i>p</i><0.05 vs. non diabetic # <i>p</i><0.01 vs. diabetes, ^$<i>p</i><0.05 vs. diabetes + CoPP values are presented as means ± SD; n = 7 in control and CoPP group; n = 5 in other groups values are presented as means ± SD).</p

HO-1 induction increases HO-1, AKT and GSK3β expression in diabetic hearts.

<p>A:CoPP treatment significantly increased HO-1 expression levels (Means ± SD, n = 5. ^$<i>p</i><0.005 vs. sham, *<i>p</i><0.01 vs. diabetes,). B: Western blot analysis of AKT and p-AKT and p-Akt/Akt ratio in the different groups. CoPP treatment increased p-Akt levels while SnPP decreased the expression of p-AKT compared to the CoPP treated diabetic mice. (Means±SD, n = 5. ^$<0.05 vs. sham,*<i>p</i><0.05 vs. diabetes, #<i>p</i><0.05 vs. diabetes+CoPP C: Western blot analysis of GSK-3β in mice subjected to LAD ligation. CoPP treatment led to an increase in GSK-3β levels; SnPP decreased GSK-3β expression compared to the CoPP treated diabetic mice (Means ± SD, n = 5. ^$<0.05 vs. sham, * <i>p</i><0.05 vs. diabetes, #<i>p</i><0.05 vs. diabetes+CoPP).</p

CoPP increases HO-1,AKT and GSK3β expression in cardiomyocytes culture.

<p>A: Western blot analysis of HO-1 protein in the cardiomyocyte subjected to hypoxia CoPP treatment increased HO-1 expression levels in cells subjected to hypoxia compared to non-treated cells subjected to hypoxia. Means ± SD, n = 3 in 3 different experiments (*<i>p</i><0.05 vs. normoxia, ^#<i>p</i><0.005 vs. hypoxia). B: Western blot analysis of AKT protein in the cardiomyocytes subjected to hypoxia: CoPP treatment increased AKT phosphorylation in cells subjected to hypoxia compared to non-treated cells subjected to hypoxia Means ± SD, n = 3 in 3 different experiments. (*<i>p</i><0.01 vs. normoxia, ^#<i>p</i><0.05 vs. hypoxia, ^{p<0.05 vs. hyp+CoPP). C: Western blot analysis of pGSK3β protein in the cardiomyocytes subjected to hypoxia CoPP treatment increased GSK3β phosphorylation in cells subjected to hypoxia compared to non-treated cells subjected to hypoxia. Means ± SD, n = 3 in 3 different experiments (*p<0.01 vs. normoxia, ^#p<0.01 vs. hypoxia,}<i>p</i><0.05 vs. hyp+CoPP).</p

Anti-apoptotic effects of HO-1 on cardiomyocytes in diabetic mice.

<p>A: Western blot analysis of Bax B: Bcl-2 C: and Bcl-2/Bax ratio, in mice subjected to LAD ligation. CoPP treatment increased the levels of Bcl-2 expression but it did not change Bax expression to shift the Bcl-2/Bax ratio towards the antiapoptotic Bcl-2 (Means ± SD, n = 5. *<i>p</i><0.05 vs. diabetes+MI, ^#<i>p</i><0.05 vs. diabetes+CoPP, Table, *<i>p</i><0.05 vs. diabetes+MI, ^#<i>p</i><0.05 vs. diabetes+CoPP).</p

Measurements of biochemical markers in CoPP-pretreated cardiomyocytes subjected to hypoxia.

<p>The release of LDH and CK after 2 h of hypoxia was lower in the group treated with CoPP compared with the untreated group. SnPP administration abolished the beneficial effect of CoPP pretreatment. The value of 100% was given to the level of LDH or CK released from untreated cells not subjected to hypoxia. Data are means of at least 3 replicates in 5 separate experiments. Values are means ± SD (LDH ^{p<0.01 vs. normoxia, ^#p<0.01 vs. hypoxia, *p<0.01 vs. hyp +CoPP, CK}<i>p</i><0.01 vs. normoxia, ^#<i>p</i><0.01 vs. hypoxia, *<i>p</i><0.01 vs. hyp +CoPP).</p

Anti-inflammatory and anti-oxidant effects of HO-1 induction on cardiomyocytes in diabetic mice.

<p>A: Superoxide levels of the cardiac tissue in mice subjected to LAD ligation. CoPP treatment increased the levels of superoxide (Means ± SD, n = 5. *<0.05 vs. non diabetes MI, #<i>p</i><0.05 vs. diabetes MI, + <i>p</i><0.05 vs. diabetes+CoPP. B,C: Plasma levels of TNFalpha and adiponectin respectively, in mice subjected to LAD ligation. (Means ± SD, n = 5. *<0.05 vs. non diabetes MI, #<i>p</i><0.05 vs. diabetes MI, + <i>p</i><0.05 vs. diabetes+CoPP MI).</p