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Heterozygous disruption of Flk-1 receptor leads to myocardial ischaemia reperfusion injury in mice: application of affymetrix gene chip analysis.

ABSTRACT: This study addresses an important clinical issue by identifying potential candidates of vascular endothelial growth factor (VEGF) signalling through the Flk-1 receptor that trigger cardioprotective signals under ischaemic stress. Isolated working mouse hearts of both wild-type (WT) and Flk-1(+/-) were subjected to global ischaemia (I) for 30 min. followed by 2 hrs of reperfusion (R). Flk-1(+/-) myocardium displayed almost 50% reduction in Flk-1 mRNA as examined by quantitative real-time RT-PCR at the baseline level. Flk-1(+/-) mouse hearts displayed reduction in left ventricular functional recovery throughout reperfusion (dp/dt 605 versus 884), after 2 hrs (P<0.05). Coronary (1.9 versus 2.4 ml) and aortic flow (AF) (0.16 versus 1.2 ml) were reduced in Flk-1(+/-) after 2 hrs of reperfusion. In addition, increased infarct size (38.4%versus 28.41%, P<0.05) and apoptotic cardiomyocytes (495 versus 213) were observed in Flk-1(+/-) knockout (KO) mice. We also examined whether ischaemic preconditioning (PC), a novel method to induce cardioprotection against ischaemia reperfusion injury, through stimulating the VEGF signalling pathway might function in Flk-1(+/-) mice. We found that knocking down Flk-1 resulted in significant reduction in the cardioprotective effect by PC compared to WT. Affymetrix gene chip analysis demonstrated down-regulation of important genes after IR and preconditioning followed by ischaemia reperfusion in Flk-1(+/-) mice compared to WT. To get insight into the underlying molecular pathways involved in ischaemic PC, we determined the distinct and overlapping biological processes using Ingenuity pathway analysis tool. Independent evidence at the mRNA level supporting the Affymetrix results were validated using real-time RT-PCR for selected down-regulated genes, which are thought to play important roles in cardioprotection after ischaemic insult. In summary, our data indicated for the first time that ischaemic PC modifies genomic responses in heterozygous VEGFR-2/Flk-1 KO mice and abolishes its cardioprotective effect on ischaemic myocardium.

SUBMITTER: Thirunavukkarasu M

PROVIDER: S-EPMC3865673 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Heterozygous disruption of Flk-1 receptor leads to myocardial ischaemia reperfusion injury in mice: application of affymetrix gene chip analysis.

Thirunavukkarasu M M Addya S S Juhasz B B Pant R R Zhan L L Surrey S S Maulik G G Menon V P VP Maulik N N

Journal of cellular and molecular medicine 20080208 4

This study addresses an important clinical issue by identifying potential candidates of vascular endothelial growth factor (VEGF) signalling through the Flk-1 receptor that trigger cardioprotective signals under ischaemic stress. Isolated working mouse hearts of both wild-type (WT) and Flk-1(+/-) were subjected to global ischaemia (I) for 30 min. followed by 2 hrs of reperfusion (R). Flk-1(+/-) myocardium displayed almost 50% reduction in Flk-1 mRNA as examined by quantitative real-time RT-PCR a ...[more]

PMID: 18266966

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Project description:Aims:Myocardial ischaemia followed by reperfusion (IR) causes an oxidative burst resulting in cellular dysfunction. Little is known about the impact of oxidative stress on cardiomyocyte lipids and their role in cardiac cell death. Our goal was to identify oxidized phosphatidylcholine-containing phospholipids (OxPL) generated during IR, and to determine their impact on cell viability and myocardial infarct size. Methods and results:OxPL were quantitated in isolated rat cardiomyocytes using mass spectrophotometry following 24 h of IR. Cardiomyocyte cell death was quantitated following exogenously added OxPL and in the absence or presence of E06, a 'natural' murine monoclonal antibody that binds to the PC headgroup of OxPL. The impact of OxPL on mitochondria in cardiomyocytes was also determined using cell fractionation and Bnip expression. Transgenic Ldlr-/- mice, overexpressing a single-chain variable fragment of E06 (Ldlr-/--E06-scFv-Tg) were used to assess the effect of inactivating endogenously generated OxPL in vivo on myocardial infarct size. Following IR in vitro, isolated rat cardiomyocytes showed a significant increase in the specific OxPLs PONPC, POVPC, PAzPC, and PGPC (P < 0.05 to P < 0.001 for all). Exogenously added OxPLs resulted in significant death of rat cardiomyocytes, an effect inhibited by E06 (percent cell death with added POVPC was 22.6 ± 4.14% and with PONPC was 25.3 ± 3.4% compared to 8.0 ± 1.6% and 6.4 ± 1.0%, respectively, with the addition of E06, P < 0.05 for both). IR increased mitochondrial content of OxPL in rat cardiomyocytes and also increased expression of Bcl-2 death protein 3 (Bnip3), which was inhibited in presence of E06. Notably cardiomyocytes with Bnip3 knock-down were protected against cytotoxic effects of OxPL. In mice exposed to myocardial IR in vivo, compared to Ldlr-/- mice, Ldlr-/--E06-scFv-Tg mice had significantly smaller myocardial infarct size normalized to area at risk (72.4 ± 21.9% vs. 47.7 ± 17.6%, P = 0.023). Conclusions:OxPL are generated within cardiomyocytes during IR and have detrimental effects on cardiomyocyte viability. Inactivation of OxPL in vivo results in a reduction of infarct size.

Project description:Research focus recently shifted to mitochondrial dynamics and the role of fusion and fission in cardioprotection. The aim of this study was to evaluate (i) the function and dynamics of mitochondria isolated from hearts exposed to ischaemia/reperfusion (I/R) (ii) the effects of melatonin, a powerful cardioprotectant, on mitochondrial dynamics in I/R. Isolated perfused rat hearts were stabilized for 30 min, subjected to 20 min global ischaemia, followed by 30 min reperfusion. Tissue was collected, mitochondria isolated for measurement of mitochondrial oxidative function and lysates from mitochondrial and cytosolic fractions prepared for western blotting. Melatonin (0.3 or 50 ?M) was administered for 10 min immediately before the onset of ischaemia and for 10 min at the onset of reperfusion. Infarct size was assessed after 35 min regional ischaemia/60 min reperfusion using triphenyltetrazolium staining. The results show that reperfusion significantly reduced mitochondrial QO2 (states 3 and 4), with minor effects by melatonin. Cytosolic Beclin 1 and the LC3 II/I ratio were reduced by ischaemia and increased by reperfusion. Both ischaemia and reperfusion reduced mitochondrial PINK1 and Parkin levels, while reperfusion increased p62. An alternative mitophagy pathway mediated by Rab9 is activated during myocardial ischaemia/reperfusion. Ischaemia reduced and reperfusion increased cytosolic ULK1 expression, associated with redistribution of Rab9 and Drp1 between the cytosol and mitochondria. Melatonin significantly reduced mitochondrial p62 expression upon reperfusion. Throughout the protocol, melatonin significantly (i) increased cytosolic total (t) and phospho (p) ULK1, and Rab9 levels (ii) increased the cytosolic and reduced the mitochondrial pDrp1 levels and p/t Drp1 ratio, suggesting inhibition of mitochondrial fission. Fusion was affected to a lesser extent. Cardioprotection by melatonin is associated with substantial effects on mitophagy, the significance thereof remains to be established.

Project description:Background and purposeThe immune system plays an important role in driving the acute inflammatory response following myocardial ischaemia/reperfusion injury (MIRI). IL-21 is a pleiotropic cytokine with multiple immunomodulatory effects, but its role in MIRI is not known.Experimental approachMyocardial injury, neutrophil infiltration and the expression of neutrophil chemokines KC (CXCL1) and MIP-2 (CXCL2) were studied in a mouse model of MIRI. Effects of IL-21 on the expression of KC and MIP-2 in neonatal mouse cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were determined by real-time PCR and ELISA. The signalling mechanisms underlying these effects were explored by western blot analysis.Key resultsIL-21 was elevated within the acute phase of murine MIRI. Neutralization of IL-21 attenuated myocardial injury, as illustrated by reduced infarct size, decreased cardiac troponin T levels and improved cardiac function, whereas exogenous IL-21 administration exerted opposite effects. IL-21 increased the infiltration of neutrophils and increased the expression of KC and MIP-2 in myocardial tissue following MIRI. Moreover, neutrophil depletion attenuated the IL-21-induced myocardial injury. Mechanistically, IL-21 increased the production of KC and MIP-2 in neonatal CMs and CFs, and enhanced neutrophil migration, as revealed by the migration assay. Furthermore, we demonstrated that this IL-21-mediated increase in chemokine expression involved the activation of Akt/NF-κB signalling in CMs and p38 MAPK/NF-κB signalling in CFs.Conclusions and implicationsOur data provide novel evidence that IL-21 plays a pathogenic role in MIRI, most likely by promoting cardiac neutrophil infiltration. Therefore, targeting IL-21 may have therapeutic potential as a treatment for MIRI.Linked articlesThis article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Project description:Brief episodes of ischaemia and reperfusion render the heart resistant to subsequent prolonged ischaemic insult, termed ischaemic preconditioning. Here, we hypothesized that transient non-ischaemic stress by hypertrophic stimulation would induce endogenous cardioprotective signalling and enhance cardiac resistance to subsequent ischaemic damage. Transient transverse aortic constriction (TAC) or Ang-Ⅱ treatment was performed for 3-7 days in male mice and then withdrawn for several days by either aortic debanding or discontinuing Ang-Ⅱ treatment, followed by subsequent exposure to regional myocardial ischaemia by in situ coronary artery ligation. Following ischaemia/reperfusion (I/R) injury, myocardial infarct size and apoptosis were markedly reduced and contractile function was significantly improved in the TAC preconditioning group compared with that in the control group. Similar results were observed in mice receiving Ang-Ⅱ infusion. Mechanistically, TAC preconditioning enhanced ALDH2 activity, promoted AMPK activation and improved mitochondrial energy metabolism by increasing myocardial OXPHOS complex expression, elevating the mitochondrial ATP content and improving viable myocardium glucose uptake. Moreover, TAC preconditioning significantly mitigated I/R-induced myocardial iNOS/gp91phox activation, inhibited endoplasmic reticulum stress and ameliorated mitochondrial impairment. Using a pharmacological approach to inhibit AMPK signalling in the presence or absence of preconditioning, we demonstrated AMPK-dependent protective mechanisms of TAC preconditioning against I/R injury. Furthermore, treatment with adenovirus-encoded ALDH2 partially emulated the actions of hypertrophic preconditioning, as evidenced by improved mitochondrial metabolism, inhibited oxidative stress-induced mitochondrial damage and attenuated cell death through an AMPK-dependent mechanism, whereas genetic ablation of ALDH2 abrogated the aforementioned actions of TAC preconditioning. The present study demonstrates that preconditioning with hypertrophic stress protects the heart from I/R injury via mechanisms that improve mitochondrial metabolism, reduce oxidative/nitrative stress and inhibit apoptosis. ALDH2 is obligatorily required for the development of cardiac hypertrophic preconditioning and acts as the mediator of this process.

Dataset Information

Heterozygous disruption of Flk-1 receptor leads to myocardial ischaemia reperfusion injury in mice: application of affymetrix gene chip analysis.

Publications

Heterozygous disruption of Flk-1 receptor leads to myocardial ischaemia reperfusion injury in mice: application of affymetrix gene chip analysis.

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