Insulin and GSK3ß-inhibition abrogates the infarct sparing-effect of ischemic postconditioning in ex vivo rat hearts.

OBJECTIVES: Pharmacological treatment of reperfusion injury using insulin and GSK3ß inhibition has been shown to be cardioprotective, however, their interaction with the endogenous cardioprotective strategy, ischemic postconditioning, is not known. DESIGN: Langendorff perfused ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. For the first 15 min of reperfusion hearts received either vehicle (Ctr), insulin (Ins) or a GSK3ß inhibitor (SB415286; SB41), with or without interruption of ischemic postconditioning (IPost; 3 × 30 s of global ischemia). In addition, the combination of insulin and SB41 for 15 min was assessed. RESULTS: Insulin, SB41 or IPost significantly reduced infarct size versus vehicle treated controls (IPost 33.5 ± 3.3%, Ins 33.5 ± 3.4%, SB41 30.5 ± 3.0% vs. Ctr 54.7 ± 6.8%, p < 0.01). Combining insulin and SB415286 did not confer additional cardioprotection compared to the treatments given alone (SB41 + Ins 26.7 ± 3.5%, ns). Conversely, combining either of the pharmacological reperfusion treatments with IPost completely abrogated the cardioprotection afforded by the treatments separately (Ins + IPost 59.5 ± 3.4% vs. Ins 33.5 ± 3.4% and SB41 + IPost 50.2 ± 6.6% vs. SB41 30.5 ± 3.0%, both p < 0.01), and was associated with blunted Akt, GSK3ß and STAT3 phosphorylation. CONCLUSION: Pharmacological reperfusion treatment with insulin and SB41 interferes with the cardioprotection afforded by ischemic postconditioning.

Intermittent insulin treatment mimics ischemic postconditioning via MitoKATP channels, ROS, and RISK.

OBJECTIVES: It has previously been demonstrated that 15-min continuous insulin infusion at immediate reperfusion affords cardioprotection. This study sought to reduce the treatment time of insulin and test if intermittent insulin infusions can mimic ischemic postconditioning. DESIGN: In a Langendorff perfused rat heart model of regional ischemia, hearts were at the onset of reperfusion subjected to either 5- or 1-min continuous insulin infusion or 3 × 30 s intermittent insulin infusions (InsPost); with or without inhibitors of Akt (SH-6), p70s6-kinase (rapamycin), mitochondrial ATP-sensitive potassium channels (5-hydroxydecanoic acid [5-HD]), or a scavenger of reactive oxygen species (ROS; 2-mercaptopropionyl glycine [MPG]). Infarct size is expressed as percent of area at risk and presented as mean ± standard error of the mean or s.e.m. RESULTS: Only InsPost was able to reduce infarct size compared with controls (InsPost 33 ± 6% vs. Ctr 52 ± 4%, p < 0.05.). This cardioprotection was abrogated by co-administering SH-6, rapamycin, 5-HD, or MPG. (InsPost + SH-6 56 ± 9%, InsPost + Rapa 55 ± 8%, InsPost + 5-HD 56 ± 7%, InsPost + MPG 60 ± 3% vs. InsPost 33 ± 6% p < 0.05). These results were corroborated by a significant increase in phosphorylated Akt and p70s6k in the InsPost group compared with controls. CONCLUSION: Short intermittent insulin infusions can mimic ischemic postconditioning and reduce myocardial infarct size via Akt/p70s6k and mKATP channels/ROS-dependent signaling.

Esmolol added in repeated, cold, oxygenated blood cardioplegia improves myocardial function after cardiopulmonary bypass.

OBJECTIVE: This study investigated if the ß-receptor blocking agent esmolol, added to standard oxygenated blood cardioplegia, improved myocardial function after weaning from bypass. DESIGN: A block-randomized, blinded study. SETTING: A university laboratory. PARTICIPANTS: Twenty anesthetized pigs, Norwegian Landrace. INTERVENTIONS: After cardiopulmonary bypass, cardiac arrest was induced with cold (12°C), oxygenated blood cardioplegia, enriched with either esmolol or vehicle, repeated every 20 minutes. After 100 minutes the heart was reperfused and weaned. MEASUREMENTS AND MAIN RESULTS: Left ventricular function was evaluated with pressure-volume loops, local myocardial function with multilayer strain and strain rate by epicardial short-axis tissue Doppler imaging. One hour after declamping, preload recruitable stroke work did not differ between groups, but increased to 72±3 mmHg in esmolol-treated animals v 57±4 mmHg (p<0.001) in controls after 3 hours. Radial peak ejection strain rate also was increased by esmolol; 6.0±1.0 s(-1)v 2.9±0.3 s(-1) (p<0.001) in subendocardium and 3.9±0.5 s(-1)v 2.3±0.2 s(-1) (p<0.005) in the midmyocardium. Cardiac index was increased, 4.0±0.2 L/min/m(2) by esmolol v 3.3±0.1 L/min/m(2) for controls (p<0.05). Isovolumetric relaxation time constant was reduced by esmolol, 23±1 ms v 26±1 ms (p<0.025). Troponin-T did not differ and was 339±48 ng/L for the esmolol group and 357±55 ng/L for the control group (p = 0.81). CONCLUSIONS: Esmolol added to blood cardioplegia preserved systolic cardiac function during the first 3 hours after reperfusion in a porcine model with 100 minutes of cardioplegic arrest.

Normalization strategy is critical for the outcome of miRNA expression analyses in the rat heart.

Since normalization strategies plays a pivotal role for obtaining reliable results when performing quantitative PCR (qPCR) analyses, this study investigated several miRNA normalization candidates in regards to their efficiency as normalization standards in the ischemic reperfused ex vivo rat heart, with special reference to regulation of the miRNAs miR-1 and miR-101b. The possibility of including primers for several miRNAs in one reverse transcription (RT) reaction was also investigated. Langendorff perfused rat hearts were subjected to 30 min regional ischemia and 0, 1, 5, 15, or 120 min reperfusion. Total RNA was isolated and reverse transcribed for miRNA qPCR analysis. Normalization candidates were evaluated by the NormFinder and geNorm algorithms and the following stability expression rank order was obtained: sno202 < U6B < U87 < snoRNA < 4.5S RNA A < Y1 < 4.5S RNA B < GAPDH. Applying U6B as a normalizer it was found that miR-1 and miR-101b was downregulated in the ischemic reperfused myocardium. Furthermore, up to three primers could be included in one RT reaction by replacing RNase-free water with two supplemental sets of primers in the TaqMan MicroRNA assay protocol. This study demonstrates the importance of validating normalization standards when performing miRNA expression analyses by qPCR, and that miR-1 and miR-101b may play an important role during early reperfusion of the ischemic rat heart.

Myasthenia gravis sera have no effect on cardiomyocytes in vitro.

Myasthenia gravis (MG) is an autoimmune disorder primarily caused by circulating autoantibodies targeting the nicotinic acetylcholine receptor. Several studies have suggested a link between MG and heart disease. Girardi heart cells were treated with MG sera, measuring cytotoxic effects using flow cytometry, adenylate kinase (AK) release and evaluating morphology. MG sera did not induce morphological changes in the cells. AK release from cells treated with MG sera did not exceed controls and flow cytometric examination did not reveal any increase in dead or apoptotic cells. We conclude that MG sera have no cytotoxic effect in our heart cell culture system.

Insulin Postconditioning Reduces Infarct Size in the Porcine Heart in a Dose-Dependent Manner.

AIM: Insulin and glucose may have opposite effects when used to reduce ischemia-reperfusion injury. When insulin is administered alone, feeding state determines tolerance and further induces metabolic and hormonal changes. Higher insulin doses are needed for similar activation of cardioprotective Akt signaling in the fed compared to the fasted pig heart. Thus, the aim of the study was to investigate the effects of 2 prespecified insulin doses on infarct size, apoptosis, metabolism, and cardiac function in a clinically relevant, randomized large animal model using conventional percutaneous catheter intervention techniques and including different fasting states. METHODS AND RESULTS: Twenty-seven female pigs were subjected to 40-minute ischemia and 120-minute reperfusion. Pharmacological postconditioning with intracoronary infusions administered over 3 × 30 seconds was performed at immediate reperfusion. Animals were randomly assigned to 3 groups-preexperimental fasting and intracoronary saline ( controls), preexperimental fasting and 0.1U of insulin ( fasted Ins0.1U), and preexperimental feeding and 1.0U of insulin ( fed Ins1.0U). A significant reduction in infarct size was demonstrated in the fed Ins1.0U group ( P = .047) but not in the fasted Ins0.1U group ( P = .531) compared to controls (infarct size normalized to area at risk ± standard deviation: controls 70.2% ± 12.9%, fasted Ins0.1U 65.0% ± 9.4%, and fed Ins1.0U 54.4% ± 7.3%). Infarct limitation was associated with more uncleaved caspase-3 in the area of risk and the infarcted area, lower circulating free fatty acids, and less increase in heart rate during reperfusion. Fed animals had higher levels of glucose, carnitine, potassium, and normetanephrine and higher heart rate at baseline compared to controls. CONCLUSION: Insulin postconditioning reduced infarct size in the in vivo pig heart, but the beneficial effects were restricted to the highest dose, which is limited by side effects and can only be given to nonfasted animals. The finding challenges successful general use of insulin in the treatment of reperfusion injury in clinical acute myocardial infarction.

Urocortin-II and urocortin-III are cardioprotective against ischemia reperfusion injury: an essential endogenous cardioprotective role for corticotropin releasing factor receptor type 2 in the murine heart.

Corticotropin-releasing factor (CRF) receptor type 2beta (CRFR2beta) is expressed in the heart. Urocortin (Ucn)-I activation of CRFR2beta is cardioprotective against ischemic reperfusion (I/R) injury by stimulation of the ERKs1/2 p42, 44. However, by binding CRF receptor type 1, Ucn-I can also activate the hypothalamic stress axis. Ucn-II/stresscopin related peptide and Ucn-III/stresscopin are two new members of the CRF/Ucn-I gene family and are selective for CRFR2beta. We propose that CRFR2beta selective Ucn-II or Ucn-III will protect cardiomyocytes and the ex vivo Langendorff perfused rat heart from I/R injury by activation of ERK1/2-p42, 44. Ucn-II is expressed in mouse cardiomyocytes, and Ucn-II or Ucn-III can bind to CRFR2beta, resulting in ERK1/2-p42, p-44 phosphorylation and cAMP stimulation. Phosphorylation of ERK1/2-p42, p-44 is regulated by the Ras/Raf-1 kinase pathway, independent of adenylate cyclase and, therefore, cAMP activation. Ucn-II and Ucn-III protect cardiomyocytes from I/R injury and reduce the percentage of infarct size:risk ratio in Langendorff perfused rat hearts exposed to regional I/R (P<0.001). The CRFR2 selective antagonist astressin2-B and an ERK1/2-p42, 44 inhibitor abolish the cardioprotective actions of Ucn-II and Ucn-III in reperfusion. Cardiomyocytes isolated from CRFR2-null mice are less resistant to I/R injury, compared with wild-type cardiomyocytes. We propose the use of CRFR2 selective agonists, Ucn-II and Ucn-III, to treat ischemic heart disease because of their potent cardioprotective effects in the murine heart and their minimal impact on the hypothalamic stress axis. We emphasize an important endogenous cardioprotective role for CRFR2beta in the murine heart.

Activation of corticotropin releasing factor receptor type 2 in the heart by corticotropin releasing factor offers cytoprotection against ischemic injury via PKA and PKC dependent signaling.

Corticotrophin-releasing factor receptor 2ß (CRFR2ß) is expressed in the myocardium. In the present study we explore whether acute treatment with the neuropeptide corticotrophin-releasing factor (CRF) could induce cytoprotection against a lethal ischemic insult in the heart (isolated murine neonatal cardiac myocytes and the isolated Langendorff perfused rat heart) by activating CRFR2. In vitro, CRF offered cytoprotection when added prior to lethal simulated ischemic stress by reducing apoptotic and necrotic cell death. Ex vivo, CRF significantly reduced infarct size from 52.1±3.1% in control hearts to 35.3±3.1% (P<0.001) when administered prior to a lethal ischemic insult. The CRF peptide did not confer cytoprotection when administered at the point of hypoxic reoxygenation or ischemic reperfusion. The acute effects of CRF treatment are mediated by CRF receptor type 2 (CRFR2) since the cardioprotection ex vivo was inhibited by the CRFR2 antagonist astressin-2B. Inhibition of the mitogen activated protein kinase-ERK1/2 by PD98059 failed to inhibit the effect of CRF. However, both protein kinase A and protein kinase C inhibitors abrogated CRF-mediated protection both ex vivo and in vitro. These data suggest that the CRF peptide reduces both apoptotic and necrotic cell death in cardiac myocytes subjected to lethal ischemic induced stress through activation of PKA and PKC dependent signaling pathways downstream of CRFR2.

Daunorubicin therapy is associated with upregulation of E3 ubiquitin ligases in the heart.

Daunorubicin (DNR) and doxorubicin (DOX) are two of the most effective anthracycline drugs known for the treatment of systemic neoplasms and solid tumors. However, their clinical use is hampered due to profound cardiotoxicity. The mechanism by which DNR injures the heart remains to be fully elucidated. Recent reports have indicated that DOX activates ubiquitin proteasome-mediated degradation of specific transcription factors; however, no reports exist on the effect of DNR on the E3 ubiquitin ligases, MURF-1 (muscle ring finger 1) and MAFbx (muscle atrophy F-box). The aim of this study was to investigate the effect of DNR treatment on the protein and organelle degradation systems in the heart and to elucidate some of the signalling mechanisms involved. Adult rats were divided into two groups where one group received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze-clamped for biochemical analysis. DNR treatment significantly attenuated cardiac function and increased apoptosis in the heart. DNR-induced cardiac cytotoxicity was associated with upregulation of the E3 ligases, MURF-1 and MAFbx and also caused significant increases in two markers of autophagy, beclin-1 and LC3. These changes observed in the heart were also associated with attenuation of the phosphoinositide 3-kinase/Akt signalling pathway.

Percutaneous catheter-based intracoronary infusion of insulin--a dose finding study in the porcine model.

Insulin given at immediate reperfusion reduces myocardial infarct size in the in vitro and the ex vivo rat heart. In vivo, insulin may cause hypoglycaemia, hypokalaemia and elevation of catecholamines, potentially harmful during an acute myocardial infarction. The purpose of this study was to evaluate tolerance and safety of intracoronary insulin infusions in a porcine model applying percutaneous intervention techniques.

Human catestatin peptides differentially regulate infarct size in the ischemic-reperfused rat heart.

In acute myocardial infarction increased plasma levels of chromogranin A are correlated with decreased survival. At the human chromogranin A gene locus there are two naturally occurring amino acid substitution variants within the catestatin region, i.e. Gly³64Ser and Pro³7°Leu, displaying differential potencies towards inhibition of nicotinic cholinergic agonist-evoked catecholamine secretion from sympathochromaffin cells and different degrees of processing from the prohormone. Here, we examine whether two of the variants and the wild type catestatin may affect the development of infarct size during ischemic reperfusion in the Langendorff rat heart model. The hearts were subjected to regional ischemia followed by reperfusion in the presence or absence of synthetic variants of human catestatin. Compared to the Gly³64Ser variant both the wild type and Pro³7°Leu variants increased infarct size while decreasing the cardiac levels of phosphorylated Akt and two of its downstream targets, FoxO1 and BAD. In conclusion, these findings suggest that, in contrast to the Gly³64Ser variant, wild type catestatin and the Pro³7°Leu variant (allele frequency ~0.3%) increased myocardial infarct size via a mechanism involving dephosphorylation of Akt and the two downstream targets during ischemic reperfusion in the isolated rat heart.

Signal transducer and activator of transcription 3 is involved in the cardioprotective signalling pathway activated by insulin therapy at reperfusion.

OBJECTIVE: To evaluate the significance of the JAK-STAT pathway in insulin-induced cardioprotection from reperfusion injury. METHODS: In isolated perfused rat hearts subjected to insulin therapy (0.3 mU/ml) +/- AG490 (5 microM, JAK-STAT inhibitor), the phosphorylation state of STAT3 and Akt was determined after 15 min of reperfusion. Infarct size was measured after 120 min of reperfusion. Isolated cardiac myocytes from wild type (WT) and cardiac specific STAT3 deficient mice were treated with insulin at reoxygenation following simulated ischemia (SI, 26 h). Cell viability was measured after 120 min of reoxygenation following SI, whereas phosphorylation state of Akt was measured after 15 min of reoxygenation following SI. RESULTS: Insulin given at reperfusion led to phosphorylation of STAT3 and Akt both of which were inhibited by AG490. AG490 also blocked the insulin-dependent decrease in infarct size, supporting a role for JAK-STAT in cardioprotection. In addition, insulin protection from SI was blocked in myocytes from the STAT3 deficient mice, or in WT mice treated with AG490. Furthermore, insulin failed to phosphorylate Akt in the STAT3 deficient cardiomyocytes. CONCLUSION: Insulin-induced cardioprotection at reperfusion occurs through activation of STAT3. Inhibiting STAT3 by AG490, or STAT3 depletion in cardiac myocytes affects activation of Akt, suggesting close interaction between STAT3 and Akt in the cardioprotective signalling pathway activated by insulin treatment at reperfusion.

Preconditioning by 17beta-estradiol in isolated rat heart depends on PI3-K/PKB pathway, PKC, and ROS.

To study the cell signaling events leading to 17beta-estradiol (E(2))-induced acute cardioprotection, we subjected isolated rat hearts to three 5-min cycles of 10 microM E(2) before 30 min of regional ischemia, followed by 2 h of reperfusion. Protection was judged by changes in infarct size in percentage of risk zone volume. To test the importance of phosphoinositide 3-kinase (PI3-K), protein kinase C (PKC), or reactive oxygen species (ROS) in E(2)-induced protection, we combined wortmannin (1 microM), chelerythrine (2 microM), and 2-mercaptopropionylglycine (300 microM), respectively, with E(2) exposure. Changes in phosphorylation of protein kinase B (PKB) and selected PKC isoforms were tested by immunoblotting of total lysates and subcellular fractions, along with assessment of PKC translocation from soluble to membrane fraction of heart tissue homogenates. Intracellular ROS levels induced by E(2) preconditioning were investigated. E(2) preconditioning led to significant reduction in infarct size from 31.8 +/- 5.3 to 20.2 +/- 2.6% in male hearts and from 42.7 +/- 4.7 to 17.1 +/- 3.4% in female hearts (P < 0.05). Protection was abolished by wortmannin (30.0 +/- 3.2%), chelerythrine (45.1 +/- 4.4%), and 2-mercaptopropionylglycine (36.8 +/- 4.7%). E(2) preconditioning induced phosphorylation of PKB, PKCalpha, and PKCepsilon and membrane translocation of PKCepsilon and PKCdelta. Intracellular ROS levels were found elevated after transient treatment with hormone. Therefore, our data demonstrate the ability of E(2) to induce preconditioning-like cardioprotection via cell signaling events shared by classic ischemic preconditioning.

p70s6 kinase is a functional target of insulin activated Akt cell-survival signaling.

Insulin administration attenuates cardiac ischemia-reperfusion apoptosis via activation of Akt-mediated cell-survival signaling. As p70s6 kinase is a cognate Akt-mediated phosphorylation target we evaluated whether p70s6 kinase activation is a functional requirement in insulin-mediated cell survival program during post-ischemic reoxygenation. Human cardiac-derived girardi cells were subjected to 6h of simulated ischemia and 2h of reoxygenation+/-insulin treatment [0.3mU/ml]. Concurrently, cells were pre-treated with anti-sense oligodeoxynucleotides (ODNs) corresponding to the initiation start-site of human p70s6 kinase mRNA. Sense ODN and scrambled ODN were used as controls. Cell viability was measured using lactate dehydrogenase (LDH) release and propidium iodide (PI) exclusion. Insulin at reoxygenation enhanced cell viability with attenuated LDH release (>or=50% , p<0.001 vs. ischemic controls) and reduced PI uptake by >or=30% vs. ischemic controls. The protection afforded by insulin was abolished by anti-sense ODN targeting p70s6 kinase, but not by the sense or scrambled ODNs. In parallel, insulin administration at reoxygenation significantly increased p70s6 kinase levels and activity compared with controls. P70s6 kinase activity was abolished by pre-treatment with anti-sense ODNs. Collectively, these data demonstrate that p70s6 kinase activation is a functional target of Akt following insulin-activated cytoprotection during ischemia-reoxygenation-induced injury.