Long-term consumption of an obesogenic high fat diet prior to ischemia-reperfusion mediates cardioprotection via Epac1-dependent signaling.

BACKGROUND: Obesity is still considered a risk factor for cardiovascular disease, although more recent knowledge also suggests obesity to be associated with reduced morbidity and mortality - the "obesity paradox". This study explores if long-term feeding of an obesogenic high fat diet renders the myocardium less susceptible to ischemic-reperfusion induced injury via Epac-dependent signaling. METHODS: Wild type (wt), Epac1 (Epac1-/-) and Epac2 (Epac2-/-) deficient mice were fed a high fat (HFD) or normal chow diet (ND) for 33 ± 1 weeks. Six experimental groups were included: (1) control wt ND (wt ND), (2) control wt HFD (wt HFD), (3) Epac1-/- mice on ND (Epac1-/-ND), (4) Epac1-/- mice on HFD (Epac1-/-HFD), (5) Epac2-/- mice on ND (Epac2-/-ND), and (6) Epac2-/- mice on HFD (Epac2-/-HFD). Isolated ex vivo mice hearts were perfused in a constant pressure Langendorff mode, and exposed to 30min of global ischemia (GI) and 60min of reperfusion. Endpoints were infarct size and functional recovery. RESULTS: All groups fed a HFD presented with significantly enhanced body weight, visceral fat content and reduced glucose clearance compared to corresponding ND groups. Although the HFD cohorts presented with an overall comparable systemic capability to clear glucose, the Epac1-/- HFD group presented with glucose levels slightly above the human diabetes criteria at the end of the intraperitoneal glucose tolerance test (ipGTT). Moreover, the HFD significantly reduced infarct size in both wild type (wt HFD 41.3 ± 5.5% vs. wt ND 58.0 ± 9.8%, p < 0.05) and Epac2-/- cohorts (Epac2-/-HFD 34.4 ± 7.2% vs. Epac2-/-ND 56.5 ± 3.8%, p < 0.05). Interestingly, however, the HFD did not reduce infarct size in Epac1-/- deficient mice hearts (Epac1-/-HFD 65.1 ± 5.1% vs. Epac1-/-ND 56.1 ± 3.5%, ns.). CONCLUSION: Epac1-dependent signaling is involved in mediating the cardioprotection afforded by long-term feeding of an obesogenic high fat diet in mice hearts.

B-type natriuretic peptide expression and cardioprotection is regulated by Akt dependent signaling at early reperfusion.

Exogenously administered B-type natriuretic peptide (BNP) has been shown to offer cardioprotection through activation of particulate guanylyl cyclase (pGC), protein kinase G (PKG) and KATP channel opening. The current study explores if cardioprotection afforded by short intermittent BNP administration involves PI3K/Akt/p70s6k dependent signaling, and whether this signaling pathway may participate in regulation of BNP mRNA expression at early reperfusion. Isolated Langendorff perfused rat hearts were subjected to 30min of regional ischemia and 120min of reperfusion (IR). Applying intermittent 3×30s infusion of BNP peptide in a postconditioning like manner (BNPPost) reduced infarct size by >50% compared to controls (BNPPost 17±2% vs. control 42±4%, p<0.001). Co-treatment with inhibitors of the PI3K/Akt/p70s6k pathway (wortmannin, SH-6 and rapamycin) completely abolished the infarct-limiting effect of BNP postconditioning (BNPPost+Wi 36±5%, BNPPost+SH-6 41±4%, BNPPost+Rap 37±6% vs. BNPPost 17±2%, p<0.001). Inhibition of natriuretic peptide receptors (NPR) by isatin also abrogated BNPPost cardioprotection (BNPPost+isatin 46±2% vs. BNPPost 17±2%, p<0.001). BNPPost also significantly phosphorylated Akt and p70s6k at early reperfusion, and Akt phosphorylation was inhibited by SH-6 and isatin. Myocardial BNP mRNA levels in the area at risk (AA) were significantly elevated at early reperfusion as compared to the non-ischemic area (ANA) (Ctr(AA) 2.7±0.5 vs. Ctr(ANA) 1.2±0.2, p<0.05) and the ischemic control tissue (Ctr(AA) 2.7±0.5 vs. ischemia 1.0±0.1, p<0.05). Additional experiments also revealed a significant higher BNP mRNA level in ischemic postconditioned (IPost) hearts as compared to ischemic controls (IPost 6.7±1.3 vs. ischemia 1.0±0.2, p<0.05), but showed no difference from controls run in parallel (Ctr 5.4±0.8). Akt inhibition by SH-6 completely abrogated this elevation (IPost 6.7±1.3 vs. IPost+SH-6 1.8±0.7, p<0.05) (Ctr 5.4±0.8 vs. SH-6 1.5±0.9, p<0.05). In conclusion, Akt dependent signaling is involved in mediating the cardioprotection afforded by intermittent BNP infusion at early reperfusion, and may also participate in regulation of reperfusion induced BNP expression.

Dietary red palm oil protects the heart against the cytotoxic effects of anthracycline.

Strong anti-neoplastic anthracyclines like daunorubicin (DNR) and doxorubicin (DOX) have high efficacy against systemic neoplasm and solid tumours. However, clinically, they cause chronic cardiomyopathy and congestive heart failure. Red palm oil (RPO) supplementation can protect the heart against ischemic injury. We therefore hypothesize that supplementation with RPO during chemotherapy may protect the heart. Control rats received a standard diet, and the experimental group received RPO in addition for 4 weeks. Each group was subsequently injected with either saline or DNR over a 12-day period towards the end of 4 weeks. Hearts were excised and perfused on a working heart system. Functional parameters were measured. Tissue samples were collected for analysis of mRNA and protein levels. DNR + RPO increased aortic output by 25% (p < 0.05) compared with DNR only. Furthermore, DNR treatment significantly reduced tissue mRNA levels of superoxide dismutase 1 (SOD1) and nitric oxide synthase 1 (NOS1) compared with untreated controls. Protein expression of SOD1 followed the same pattern as mRNA levels. NOS1 protein levels were significantly increased in DNR treated rats when compared with untreated controls. In addition, DNR increased phosphorylation of p38 and Jun N-terminal kinase compared with untreated controls, whereas DNR + RPO completely counteracted this activation. DNR + RPO significantly up regulated the protein extracellular signal-regulated kinase 1 level compared with DNR only. In this model of DNR treatment, RPO is associated with stabilization of important antioxidant enzymes such NOS and SOD, and inhibition of the 'stress' induced mitogen-activated protein kinase pathways. Dietary RPO also maintained function, similar to control, in DNR treated hearts.

Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion.

Short non-lethal ischemic episodes administered to hearts prior to (ischemic preconditioning, IPC) or directly after (ischemic postconditioning, IPost) ischemic events facilitate myocardial protection. Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults. We propose that coronary IPC effluent contains hydrophobic cytoprotective mediators acting via PI3K/Akt-dependent pro-survival signaling at ischemic reperfusion. Ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. IPC effluent administered for 10 min prior to index ischemia attenuated infarct size by ≥55% versus control hearts (P < 0.05). Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05). The IPC effluent significantly increased Akt phosphorylation in un-preconditioned hearts when administered before ischemia or at reperfusion, while pharmacological inhibition of PI3K/Akt-signaling at reperfusion completely abrogated the cardioprotection offered by effluent administration. Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05). The total hydrophobic effluent fraction significantly reduced infarct size independently of temporal administration (before ischemia, at reperfusion or as remote postconditioning). In conclusion, the IPC effluent retains strong cardioprotective properties, containing hydrophobic mediator(s) < 30 kDa offering cytoprotection via PI3K/Akt-dependent signaling at ischemic reperfusion.

Pretreatment with insulin before ischaemia reduces infarct size in Langendorff-perfused rat hearts.

AIM: To compare the possible role of Akt and mammalian target of rapamycin (mTOR) in mediating cardioprotection against ischaemia under three different conditions: (1) During ischaemic preconditioning (IPC), (2) when insulin was given as a pretreatment agent (InsPC) and (3) when insulin was given as a reperfusion cell survival agent (Ins(R)). METHODS: Isolated perfused rat hearts were subjected to IPC (3 x 5 min) or InsPC (50 mU mL(-1); 3 x 5 min), before 30 min of regional ischaemia followed by 120 min of reperfusion +/- 1L-6-hydroxymethyl-chiro-inositol-2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (HIMO) (20 microm; Akt inhibitor) or rapamycin (1 nm; mTOR inhibitor). In addition, insulin (3 mU mL(-1)) was given at the onset of reperfusion, +/- HIMO or rapamycin. Risk zone (R) and infarct size (I) were determined with Evans blue and tetrazolium staining respectively. Western blot analysis was performed on tissue from Langendorff-perfused rat hearts and cell lysates from cultured HL1 cells. RESULTS: IPC, InsPC and InsR treatment resulted in a significant reduction in infarct size compared to controls (all P < 0.05). This protective effect of IPC and insulin was abolished by the inhibitors. However, the putative Akt inhibitor, although capable of abolishing cardioprotection induced by insulin, was not able to inhibit insulin-induced phosphorylation of Akt in Langendorff-perfused rat hearts and cultured HL1 cells. The target for this compound therefore remains to be determined. CONCLUSION: IPC and insulin (either as InsPC or Ins(R)) appear to activate mTOR, and this kinase seems to play an essential role in cardioprotection against ischaemia and reperfusion injury as rapamycin blocked the protection.

Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling.

The "metabolic cocktail" comprising glucose-insulin-potassium administrated at reperfusion reduces infarct size in the in vivo rat heart. We propose that insulin is the major component mediating this protection and acts via Akt prosurvival signaling. This hypothesis was studied in isolated perfused rat hearts (measuring infarct size to area of risk [%]) subjected to 35 minutes regional myocardial ischemia and 2 hours reperfusion. Insulin administered at the onset of reperfusion attenuated infarct size by >/=45% versus control hearts (P<0.001). Insulin-mediated cardioprotection was found to be independent of the presence of glucose at reperfusion. Moreover, the cell survival benefit of insulin is temporally dependent, in that insulin administration from the onset of reperfusion and maintained for either 15 minutes or for the duration of reperfusion reduced infarct size. In contrast, protection was abrogated if insulin administration was delayed until 15 minutes into reperfusion. Pharmacological inhibition of both upstream and downstream signals in the Akt prosurvival pathway abolished the cardioprotective effects of insulin. Here coadministration of insulin with the tyrosine kinase inhibitor lavendustin A, the phosphatidylinositol3-kinase (PI3-kinase) inhibitor wortmannin, and mTOR/p70s6 kinase inhibitor rapamycin abolished cardioprotection. Steady-state levels of activated/phosphorylated Akt correlated with insulin administration. Finally, downstream prosurvival targets of Akt including p70s6 kinase and BAD were modulated by insulin. In conclusion, insulin administration at reperfusion reduces myocardial infarction, is dependent on early administration during reperfusion, and is mediated via Akt and p70s6 kinase dependent signaling pathway. Moreover, BAD is maintained in its inert phosphorylated state in response to insulin therapy.

5-HD abolishes ischemic preconditioning independently of monophasic action potential duration in the heart.

OBJECTIVE: Blocking of the KATP channel with either glibenclamide or 5-hydroxydecanoate (5-HD) has been shown to abolish the infarct reducing effect of ischemic preconditioning (IPC) in hearts from several species, but the results in rat and rabbit have been equivocal. In this study we investigated if 5-HD could abolish IPC in rat and rabbit and further if IPC or IPC + 5-HD were affecting action potential duration in the rabbit heart. METHODS: The rat hearts were isolated and retrogradely perfused on a Langendorff perfusion apparatus with Krebs-Henseleit buffer. The rabbit experiments were performed in an in situ model. Rat and rabbit hearts were subjected to 30 min regional ischemia by ligating a coronary artery followed by 120 min (rat) or 150 min (rabbit) of reperfusion. The preconditioning protocol was one or three cycles of 5 min ischemia plus 5 min reperfusion in the rat and one cycle of 5 min ischemia plus 10 min reperfusion in the rabbit. In the rat 5-HD was added to the reservoir before ischemic preconditioning in different concentrations, and in the rabbit 5-HD was given as a bolus 5 mg/kg intraventricularly 2 min before the preconditioning ischemia. In the rabbit epicardial monophasic action potential duration at 50 % repolarization (MAPD50) was measured at 1, 2 and 5 min in each of the ischemic periods using a contact pressure electrode. Infarcts were measured with tetrazolium staining and risk zone volumes with fluorescent microspheres. RESULTS: All data are presented as infarct size in % of risk zone volume (mean +/- SEM). In the rat 200 microM of 5-HD abolished the protective effect of one cycle of IPC (28.6 +/- 4.7 versus 8.4 +/- 0.8) and 500M of 5-HD abolished three cycles of IPC (50.7 +/- 7.8 versus 8.4 +/- 2.0). Control was 40.9 +/- 2.8. In the rabbit 5-HD abolished IPC (41.2 +/- 7.2 versus 8.1 +/- 3.2). Control was 53.5 +/- 12.4. MAPD50 were significantly more shortened compared to control at 1 and 2 min into the 30 min ischemia for the IPC and IPC+5-HD. CONCLUSIONS: We conclude that 5-HD abolishes ischemic preconditioning when given before the preconditioning ischemia in both rat and rabbit but does not abolish the ischemia induced shortening of the action potential duration in the rabbit; thus, a role for the mitochondrial KATP channel and not the sarcolemmal KATP channel in the protective mechanism behind IPC is probable.

Adenosine A(1) receptor activation induces delayed preconditioning in rats mediated by manganese superoxide dismutase.

BACKGROUND: We have previously described a second window of protection against infarction in rabbits 24 to 72 hours after adenosine A(1) receptor (A(1)R) activation. In this study, we examined the potential role of the mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD) as a potential end effector in mediating this protection. METHODS AND RESULTS: Rats were treated with an intravenous bolus of the A(1)R agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA, 75 microg/kg) or saline vehicle. They were also given a 5 mg/kg IV infusion of a 22-mer phosphorothioate oligodeoxynucleotide (ODN) with sequence antisense to the initiation site of rat Mn-SOD mRNA. Sense ODN and scrambled ODN were used as controls. Twenty-four hours later, hearts were isolated and perfused with buffer at constant pressure and subjected to 35 minutes of regional ischemia and 2 hours of reperfusion. Treatment with CCPA compared with saline vehicle (control) significantly reduced infarct size, expressed as percentage of myocardium at risk (22.3+/-3.3% versus 42.1+/-3.8%, respectively; P=0.001). This protection was completely abolished by prior treatment with antisense ODN, which had no effect on its own. Neither sense ODN nor scrambled ODN had an effect on the CCPA-induced delayed cardioprotection. In separate animals, 24 hours after the same treatment, hearts were assayed for Mn-SOD content and activity. CCPA treatment induced a significant increase in myocardial Mn-SOD content and activity compared with the control condition; this increase was abolished by pretreatment with antisense ODN. CONCLUSIONS: This is the first study to show that transient A(1)R activation induces delayed cardioprotection in the rat. These results strongly suggest an important role for mitochondrial Mn-SOD as a potential end effector of this protection.

Insulin administered at reoxygenation exerts a cardioprotective effect in myocytes by a possible anti-apoptotic mechanism.

The metabolic cocktail of glucose-insulin-potassium (GIK) has been shown to reduce mortality in humans and reduce infarct size in the rat when administered from the onset of reperfusion following an ischemic insult. The mechanisms underlying GIK mediated cardioprotection are, however, still unclear. Recent data implicates insulin "alone" as the major protagonist of cardioprotection when administered at the time of reperfusion. We have therefore begun to investigate an insulin activated signalling pathway and the putative role of apoptosis in this insulin-induced cardioprotection. Simulated ischemia and reoxygenation were induced in rat neonatal cardiocyte experiments. The administration of insulin [0.3 mU/ml] at the moment of reoxygenation (Ins(R)) enhanced myocardial cell viablility as assessed by trypan blue exclusion compared to vehicle alone treated control myocytes (Ins(R)50+/-2%v controls 70+/-1%, P<0.001). This insulin-mediated cardioprotection was due, in part to a reduction in myocyte apoptosis as measured by TUNEL (Ins(R)29+/-2%v controls 49+/-3%, P<0.001) and Annexin V staining (Ins(R)34+/-2%v controls 65+/-3%, P<0.001). These cardioprotective and anti-apoptotic effects of insulin were completely abolished by the tyrosine kinase inhibitor lavendustin A and by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin. Thus, we conclude that the early administration of insulin appears to be an effective modality to reduce reoxgygenation injury in cardiocytes, in part, via the attenuation of ischemia/reoxygenation-induced apoptosis. Moreover, the cardioprotective and anti-apoptotic effects of insulin are mediated via tyrosine kinase and PI3-kinase signalling pathways.

Ischemia-induced STAT-1 expression and activation play a critical role in cardiomyocyte apoptosis.

We show here that exposure of cardiac cells to simulated ischemia results in apoptosis and is accompanied by phosphorylation and increased expression and transcriptional activity of STAT-1. Similarly, interferon-gamma, which is known to induce STAT-1 activation, also induced apoptosis in cardiac cells. STAT-1-transfected cells were more susceptible to ischemia-induced cell death than cells transfected with a control plasmid lacking the STAT-1 coding sequence. Furthermore, an antisense STAT-1 vector reduced both ischemia- and overexpressed STAT-1-induced cell death in cardiac cells. Both STAT-1 overexpression and interferon-gamma treatment or exposure to ischemia activated the promoter of the pro-apoptotic caspase-1 gene in cardiomyocytes. Finally, ischemia/reperfusion also induced STAT-1 activation and caspase-1 processing in ventricular myocytes in the intact heart ex vivo. Immunofluorescent staining demonstrated an increase in STAT-1-positive staining in cardiomyocytes in response to ischemia/reperfusion that co-localized with terminal deoxynucleotidyl transferase dVTP nick end-labeling-positive apoptotic cells. These results suggest that STAT-1 plays a critical role in the regulation of ischemia/reperfusion-induced apoptosis in cardiac cells, acting at least in part via a caspase-1 activation-dependent pathway.

Coordinate regulation of metabolic enzyme encoding genes during cardiac development and following carvedilol therapy in spontaneously hypertensive rats.

Fuel substrate utilization is highly regulated during cardiac development and with the onset of cardiac hypertrophy. Glucose and lactate are the predominant fuel substrates utilized during cardiac development. Postnatally, a switch occurs so that fatty acids become the chief energy substrate in the nonfed adult mammalian heart. A reversion back towards fetal energy metabolism occurs with the development of cardiac hypertrophy. To evaluate the role of this substrate preference switch in the development of cardiac hypertrophy, the molecular regulation directing these switches is being explored. Thus, we have begun by defining the temporal expression patterns of genes encoding key rate-controlling enzymes directing major fuel substrate metabolism during cardiac development, with pressure-overload-induced cardiac hypertrophy, and following antihypertensive therapy in spontaneously hypertensive rats. The genes encoding the fatty acid and adult enriched rate-controlling glycolytic enzymes are expressed at low levels in the fetal and neonatal rat heart. The genes encoding these enzymes are significantly and coordinately upregulated (> or = 70%) in adult rat hearts compared to the fetal expression patterns. A reciprocal and coordinate downregulation (> or = 40% reduction) of the fatty acid and adult enriched glycolytic enzyme encoding genes are observed with the induction of pressure-overload-induced hypertrophy in spontaneously hypertensive rats compared to Wistar-Furth normotensive control rats. Antihypertensive therapy with carvedilol, a vasodilating alpha- and beta-adrenoreceptor antagonist, attenuates this reversion of the metabolic gene expression pattern towards fetal levels compared to placebo-treated littermate controls. This coordinate developmental and hypertrophy-induced regulation of genes that encode enzymes controlling both fatty acid and glycolytic catabolic pathways in the heart implicates potential mutual/overlapping regulatory signaling proteins within their gene regulatory programs. These gene regulatory pathways need to be identified and modulated in order to characterize the functional role of fuel substrate metabolism in cardiac development and with the induction of cardiac hypertrophy.

Urocortin protects against ischemic and reperfusion injury via a MAPK-dependent pathway.

Urocortin (UCN) is a peptide related to hypothalamic corticotrophin-releasing hormone and binds with high affinity to corticotrophin-releasing hormone receptor-2beta, which is expressed in the heart. In this study, we report that UCN prevented cell death when administered to primary cardiac myocyte cultures both prior to simulated hypoxia/ischemia and at the point of reoxygenation after simulated hypoxia/ischemia. UCN-mediated cell survival was measured by trypan blue exclusion, 3'-OH end labeling of DNA (TUNEL), annexin V, and fluorescence-activated cell sorting. To explore the mechanisms that could be responsible for this effect, we investigated the involvement of MAPK-dependent pathways. UCN caused rapid phosphorylation of ERK1/2-p42/44, and PD98059, which blocks the MEK1-ERK1/2-p42/44 cascade, also inhibited the survival-promoting effect of UCN. Most important, UCN reduced damage in isolated rat hearts ex vivo subjected to regional ischemia/reperfusion, with the protective effect being observed when UCN was given either prior to ischemia or at the time of reperfusion after ischemia. This suggests a novel function of UCN as a cardioprotective agent that could act when given after ischemia, at reperfusion.

Glucose-insulin-potassium reduces infarct size when administered during reperfusion.

Coronary reperfusion improves ventricular function and survival after infarction, but the metabolic conditions at this time may not be optimal to protect the heart. The objective of this study was to evaluate if metabolic support with glucose-insulin-potassium (GIK) administered at the time of coronary reperfusion could elicit the same cardioprotection as GIK infusion during the entire ischemia/reperfusion period. Three groups of anesthetized, open-chest rats were subjected to 30 minutes of regional ischemia and 180 minutes of reperfusion. Groups 1 (controls) and 2 (GIK(IR)) received saline or GIK, respectively, throughout the whole experimental period, whereas a third group (GIK(R)) received GIK from the onset of reperfusion only. Infarct size was significantly reduced in the GIK-treated groups, compared with controls (GIK(IR) 44 +/- 5% and GIK(R) 45 +/- 5% vs. control 66 +/- 4%; P < 0.05). Postischemic recovery of cardiac function improved when GIK was only administered during the reperfusion phase. Furthermore, infusion of GIK resulted in reduced plasma concentrations of free fatty acids and increased plasma glucose (both P < 0.05) compared with controls. This study demonstrates that glucose-insulin-potassium administration at the onset of the postischemic reperfusion period is as cardioprotective as administration of GIK during the entire ischemia/reperfusion period.