The cardioprotective effect of S. africana caerulea/Blue Sage in ischaemia and reperfusion induced oxidative stress.

Background: Since antiquity, alternative herbal remedies, such as S. africana caerulea/Blue Sage (BLS) water infusion extract (WIE) has been used by traditional healers, for the effective treatment of various chronic inflammatory disorders associated with reduced cellular antioxidant defense mechanisms and free radical cellular damage. In the heart, ischaemia-reperfusion (I/R) induced oxidative stress becomes an early crucial event in the pathogenesis of ischaemia-reperfusion injury (I/RI) and subsequent heart failure. Purpose/Aim: To investigate whether BLS WIE treatment during ischaemia and/or reperfusion may be cardioprotective. Study design: Isolated perfused rat hearts were exposed to 35 min regional ischaemia (RI) and 60 min reperfusion. The BLS WIE was applied: i) for the last 10 min of RI (PerT) or ii) from onset of reperfusion (PostT) or iii) both (PerT) + (PostT). Methods: Endpoints were functional recovery and infarct size (IS). In another set of experiments, left ventricles were freeze-clamped after RI and 10 min reperfusion for detection of total and phosphorylated p-ERK p44/p42, p-Akt, p-p38-MAPK, p-JNK, Nrf-2, NF-kB, Bax, Bcl-2, Caspase-3, and PGC-1α by Western blot analysis. Results: BLS (PostT) significantly increased ERK p44, p-Akt, Nrf-2, and Bcl-2 levels; significantly decreased p-p38-MAPK as well as p-JNK p46 phosphorylation; did not affect Bax levels and significantly decreased Bax/Bcl-2 ratios. This was associated with significantly reduced Caspase-3 levels and increased PGC-1α phosphorylation, particlarly when BLS WIE was administered as PostT. Conclusion: The administration of polyphenol-rich BLS WIE at different stages of ischaemia and/or reperfusion, activate/inhibit several signaling events simultaneously and mediate cardioprotection in a multitarget manner.

Long-chain free fatty acids inhibit ischaemic preconditioning of the isolated rat heart.

We recently reported that non-preconditioned hearts from diet-induced obese rats showed, compared to controls, a significant reduction in infarct size after ischaemia/reperfusion, whilst ischaemic preconditioning was without effect. In view of the high circulating FFA concentration in diet rats, the aims of the present study were to: (i) compare the effect of palmitate on the preconditioning potential of hearts from age-matched controls and diet rats (ii) elucidate the effects of substrate manipulation on ischaemic preconditioning. Substrate manipulation was done with dichloroacetate (DCA), which enhances glucose oxidation and decreases fatty acid oxidation. Isolated hearts from diet rats, age-matched controls or young rats, were perfused in the working mode using the following substrates: glucose (10 mM); palmitate (1.2 mM)/3% albumin) + glucose (10 mM) (HiFA + G); palmitate (1.2 mM/3% albumin) (HiFA); palmitate (0.4 mM/3% albumin) + glucose(10 mM) (LoFA + G); palmitate (0.4 mM/3% albumin) (LoFA). Hearts were preconditioned with 3 × 5 min ischaemia/reperfusion, followed by 35 min coronary ligation and 60 min reperfusion for infarct size determination (tetrazolium method) or 20 min global ischaemia/10 or 30 min reperfusion for Western blotting (ERKp44/42, PKB/Akt). Preconditioning of glucose-perfused hearts from age-matched control (but not diet) rats reduced infarct size, activated ERKp44/42 and PKB/Akt and improved functional recovery during reperfusion (ii) perfusion with HiFA + G abolished preconditioning and activation of ERKp44/42 (iii) DCA pretreatment largely reversed the harmful effects of HiFA. Hearts from non-preconditioned diet rats exhibited smaller infarcts, but could not be preconditioned, regardless of the substrate. Similar results were obtained upon substrate manipulation of hearts from young rats. Abolishment of preconditioning in diet rats may be due to altered myocardial metabolic patterns resulting from changes in circulating FA. The harmful effects of HiFA were attenuated by stimulation of glycolysis and inhibition of FA oxidation.

Antiretroviral drug-induced endothelial dysfunction is improved by dual PPARα/γ stimulation in obesity.

Obesity rates are rising in HIV-infected populations; however, the putative role of highly active antiretroviral therapy (HAART) in the development of endothelial and cardiovascular derangements in the presence of pre-existing overweight/obesity is unclear. Although dual peroxisome proliferator-activated receptors-alpha/gamma (PPARα/γ) stimulation mitigates HAART-induced metabolic dysfunction, vascular effects are unresolved. To investigate whether HAART induces vascular dysfunction in obesity and to explore the underlying mechanisms of PPARα/γ stimulation, male Wistar rats were placed on a high-calorie diet for 16 weeks. After 10 weeks, HAART (lopinavir/ritonavir, azidothymidine/lamivudine) with/without PPARα/γ agonist, Saroglitazar, was administered daily for six weeks. Excised thoracic aorta rings were subjected to isometric tension studies and Western blot measurements. HAART+Saroglitazar-treated obese animals recorded lower adiposity indices (4.3 ±â€¯0.5%) vs. HAART only-treated obese rats (5.6 ±â€¯0.3%; p < .01). Maximum acetylcholine-induced vasorelaxation (Rmax), was lower in obese+HAART group (76.10 ±â€¯3.58%) vs. obese control (101.40 ±â€¯4.75%; p < .01). However, Rmax was improved in obese+ HAART+Saroglitazar (101.00 ±â€¯3.12%) vs. obese+HAART rats (p < .001). The mean LogEC50 was improved in obese+HAART+Saroglitazar vs. obese+HAART group; p = .003. Improved endothelial function in obese+ HAART+Saroglitazar group was associated with upregulation of eNOS, PKB/Akt and downregulated p22-phox expression vs. obese+HAART group. Therefore, PPARα/γ stimulation attenuated HAART-induced endothelial dysfunction by upregulating vasoprotective eNOS, PKB/Akt signaling and downregulating pro-oxidative p22-phox expression.

Cardioprotective Effects of Beta3-Adrenergic Receptor (ß3-AR) Pre-, Per-, and Post-treatment in Ischemia-Reperfusion.

The ß3-AR (beta3-adrenergic receptor) is resistant to short-term agonist-promoted desensitization and delivers a constant intracellular signal, making this receptor a potential target in acute myocardial infarction (AMI). AIM: To investigate whether selective modulation of ß3-AR prior to or during ischemia and/or reperfusion may be cardioprotective. METHODS: Isolated perfused rat hearts were exposed to 35-min regional ischemia (RI) and 60-min reperfusion. The ß3-AR agonist (BRL37344, 1 µM) or antagonist (SR59230A, 0.1 µM) was applied: (i) before RI (PreT) or (ii) last 10 min of RI (PerT) or (iii) onset of reperfusion (PostT) or (iv) during both PerT+PostT. Nitric oxide (NO) involvement was assessed, using the NOS inhibitor, L-NAME (50 µM). Endpoints were functional recovery, infarct size (IS), cGMP levels, and Western blot analysis of eNOS, ERKp44/p42, PKB/Akt, and glycogen synthase kinase-3ß (GSK-3ß). RESULTS: Selective treatment with BRL significantly reduced IS. L-NAME abolished BRL-mediated cardioprotection. BRL (PreT) and BRL (PerT) significantly increased cGMP levels (which were reduced by L-NAME) and PKB/Akt phosphorylation. BRL (PostT) produced significantly increased cGMP levels, PKB/Akt, and ERKp44/p42 phosphorylation. BRL (PerT+PostT) caused significant eNOS, PKB/Akt, ERKp44/p42, and GSK-3ß phosphorylation. CONCLUSION: ß3-AR activation by BRL37344 induced significant cardioprotection regardless of the experimental protocol. However, the pattern of intracellular signaling with each BRL treatment differed to some degree and suggests the involvement of cGMP, eNOS, ERK, GSK-3ß, and particularly PKB/Akt activation. The data also suggest that clinical application of ß3-AR stimulation should preferably be incorporated during late ischemia or/and early reperfusion.

The significance of the washout period in preconditioning.

AIMS: Exposure of the heart to 5 min global ischaemia (I) followed by 5 min reperfusion (R) (ischaemic preconditioning, IPC) or transient Beta 2-adrenergic receptor (B2-AR) stimulation with formoterol (B2PC), followed by 5 min washout before index ischaemia, elicits cardioprotection against subsequent sustained ischaemia. As the washout period during preconditioning is essential for subsequent cardioprotection, the aim of this study was to investigate the involvement of protein kinase A (PKA), reactive oxygen species (ROS), extracellular signal-regulated kinase (ERK), PKB/Akt, p38 MAPK and c-jun N-terminal kinase (JNK) during this period. METHODS: Isolated perfused rat hearts were exposed to IPC (1x5min I / 5min R) or B2PC (1x5min Formoterol / 5min R) followed by 35 min regional ischaemia and reperfusion. Inhibitors for PKA (Rp-8CPT-cAMP)(16µM), ROS (NAC)(300µM), PKB (A-6730)(2.5µM), ERKp44/p42 (PD98,059)(10µM), p38MAPK (SB239063)(1µM) or JNK (SP600125)(10µM) were administered for 5 minutes before 5 minutes global ischaemia / 5 min reperfusion (IPC) or for 5 minutes before and during administration of formoterol (B2PC) prior to regional ischaemia, reperfusion and infarct size (IS) determination. Hearts exposed to B2PC or IPC were freeze-clamped during the washout period for Western blots analysis of PKB, ERKp44/p42, p38MAPK and JNK. RESULTS: The PKA blocker abolished both B2PC and IPC, while NAC significantly increased IS of IPC but not of B2PC. Western blot analysis showed that ERKp44/p42 and PKB activation during washout after B2PC compared to IPC was significantly increased. IPC compared to B2PC showed significant p38MAPK and JNKp54/p46 activation. PKB and ERK inhibition or p38MAPK and JNK inhibition during the washout period of B2PC and IPC respectively, significantly increased IS. CONCLUSION: PKA activation before regional ischaemia is a prerequisite for cardioprotection in both B2PC and IPC. However, ROS was crucial only in IPC. Kinase activation during the washout phase of IPC and B2PC, albeit different, affords the same cardioprotective response.

High carbohydrate and high fat diets protect the heart against ischaemia/reperfusion injury.

BACKGROUND: Although obesity is still considered a risk factor in the development of cardiovascular disorders, recent studies suggested that it may also be associated with reduced morbidity and mortality, the so-called "obesity paradox". Experimental data on the impact of diabetes, obesity and insulin resistance on myocardial ischaemia/reperfusion injury are controversial. Similar conflicting data have been reported regarding the effects of ischaemic preconditioning on ischaemia/reperfusion injury in hearts from such animals. The aim of the present study was to evaluate the susceptibility to myocardial ischaemia/reperfusion damage in two models of diet-induced obesity as well as the effect of ischaemic and pharmacological preconditioning on such hearts. METHODS: Three groups of rats were fed with: (i) normal rat chow (controls) (ii) a sucrose-supplemented diet (DIO) (iii) a high fat diet (HFD). After 16 weeks, rats were sacrificed and isolated hearts perfused in the working mode and subjected to 35 min regional ischaemia/60 min reperfusion. Endpoints were infarct size and functional recovery. Infarct size was determined, using tetrazolium staining. Activation of PKB/Akt and ERKp44/p42 (RISK pathway) during early reperfusion was determined using Western blot. Statistical evaluation was done using ANOVA and the Bonferroni correction. RESULTS: Infarct sizes of non-preconditioned hearts from the two obese groups were significantly smaller than those of the age-matched controls. Ischaemic as well as pharmacological (beta-adrenergic) preconditioning with a beta2-adrenergic receptor agonist, formoterol, caused a significant reduction in infarct size of the controls, but were without effect on infarct size of hearts from the obese groups. However, ischaemic as well as beta-preconditioning caused an improvement in functional performance during reperfusion in all three groups. A clear-cut correlation between the reduction in infarct size and activation of ERKp44/p42 and PKB/Akt was not observed: The reduction in infarct size observed in the non-preconditioned hearts from the obese groups was not associated with activation of the RISK pathway. However, beta-adrenergic preconditioning caused a significant activation of ERKp44/p42, but not PKB/Akt, in all three groups. CONCLUSIONS: Relatively long-term administration of the two obesity-inducing diets resulted in cardioprotection against ischaemia/reperfusion damage. Further protection by preconditioning was, however, without effect on infarct size, while an improvement in functional recovery was observed.

The mechanism of beta-adrenergic preconditioning: roles for adenosine and ROS during triggering and mediation.

The aim of this study was to investigate the mechanism of beta-adrenergic preconditioning (BPC). The roles of adenosine and its receptor subtypes, the generation of oxygen free radicals (ROS) and activation of the K(ATP) channels as well as the phosphoinositide-3-kinase (PI(3)K)/PKB/Akt and extracellular signal-regulated kinase (ERK) signal transduction pathways during the triggering and mediation phases were evaluated. Using the isolated working rat heart, BPC was elicited by administration of denopamine (beta1 adrenergic receptor agonist, 10(-7) M), isoproterenol (beta1/beta2 adrenergic receptor agonist, 10(-7) M) or formoterol (beta2 adrenergic receptor agonist, 10(-9) M) for 5 min followed by 5 min washout. Index ischaemia was 35 min regional ischaemia and infarct size determined using the tetrazolium method. The role of adenosine was studied using adenosine deaminase and selective antagonists as well as the PI(3)K and ERK inhibitors, wortmannin and PD98,059, bracketing the triggering and mediating phases. Involvement of ROS, PKC, the mitochondrial K(ATP) channels, release of endogenous opioids and bradykinin was studied by administration of N-acetyl cysteine (NAC), bisindolylmaleimide, the K(ATP) channel blocker 5-hydroxydecanoate (5-HD), naloxone or HOE140, respectively. Activation of PKB/Akt and ERKp44/p42 during triggering and reperfusion was determined by Western blot. Preconditioning with all three beta-adrenergic receptor agonists caused a reduction in infarct size and an improvement in postischaemic function. BPC preconditioning with isoproterenol, denopamine or formoterol was abolished by the adenosine A3 receptor antagonist MRS1191 during both the triggering and mediation phases. Isoproterenol-induced preconditioning (beta1/beta2 PC) was attenuated by MRS1754, an adenosine A(2B) receptor antagonist, during the triggering phase and abolished during reperfusion. The mediation phase of beta1/beta2 PC was also abolished by ZM241385, an adenosine A(2A) antagonist. The free radical scavenger NAC caused a significant attenuation of cardioprotection induced by isoproterenol when administered during both trigger and mediation phases, while being effective during the trigger phase with denopamine and during reperfusion in formoterol preconditioned hearts. The mitochondrial K(ATP) channel blocker, 5-HD, was without effect on beta1/beta2 PC during both triggering and mediation phases. BPC in rat hearts is dependent on activation of the A(3) adenosine receptors by endogenously produced adenosine and production of free radicals during the triggering and mediation phases while the A(2A) and A(2B) adenosine receptors participate mainly during reperfusion. The mitochondrial K(ATP) channels do not contribute to cardioprotection at any stage. Activation of ERK and PI3K/PKB/Akt during the triggering and reperfusion phases is associated with cardioprotection.

The role of ß-adrenergic receptors in the cardioprotective effects of beta-preconditioning (ßPC).

AIM: To determine the mechanism whereby transient stimulation of the ß-adrenergic receptor subtypes (ß-AR) elicit cardioprotection against subsequent ischaemia. METHODS: Isolated rat hearts were subjected to 35 min regional ischaemia (RI) and reperfusion and infarct size (IS) determined. Hearts were preconditioned with 5 min isoproterenol (ß1/ß2-AR agonist), denopamine (ß1-AR agonist), formoterol hemifumarate (ß2-AR agonist) or BRL37344 (ß3-AR agonist) and 5 min reperfusion. The roles of the ß-ARs, NO, PKA, and PI3-K were explored by using selective antagonists/blockers. Pertussis toxin was administered i.p., 48 h prior to experimentation. RESULTS: IS of hearts preconditioned with either isoproterenol, denopamine or formoterol (% of area at risk: 23.6 ± 1.26; 24.52 ± 0.89; 20.74 ± 0.85 respectively) were significantly smaller than that of non-preconditioned hearts (41.7 ± 1.65) and associated with improvement in postischaemic mechanical performance. The ß3-AR agonist BRL37344 could not reduce IS. The ß1- and ß2-AR blockers CGP-20712A and ICI-118551 abolished the reduction in IS and improvement in mechanical recovery during reperfusion induced by isoproterenol preconditioning, while the ß3-AR blocker SR59230A was without effect. Both Rp-8-CPT-cAMPs and wortmannin significantly increased IS when administered before and during ß1/ß2-AR preconditioning and reduced mechanical recovery. PTX pretreatment had no significant effect on the reduction in IS induced by ß1/ß2-AR or ß2-AR preconditioning, but reduced mechanical recovery in ß2-AR preconditioning. Similarly the NOS inhibitors L-NAME and LNNA had no effect on IS in ß1/ß2-AR preconditioning, but depressed mechanical recovery. CONCLUSION: Protection afforded by ß-ARs stimulation, depends on activation of both ß1-AR and ß2-ARs but not ß3-AR. With functional recovery as endpoint, results suggest involvement of NO in ß1/ß2-AR preconditioning and the Gi protein in ß2-AR preconditioning. Both PKA and PI3-K activation were essential for ß1/ß2-AR cardioprotection.

Effect of sildenafil on reperfusion function, infarct size, and cyclic nucleotide levels in the isolated rat heart model.

UNLABELLED: We have previously shown that NO-donor induced elevation in myocardial cGMP levels is associated with improved reperfusion function of the isolated rat heart. The phosphodiesterase 5 (PDE 5) inhibitor, sildenafil could potentially increase myocardial cGMP levels and thus protect the heart against ischaemic/reperfusion injury. METHODS: To test our hypothesis we treated the isolated working rat heart with vehicle, OR sildenafil (10, 20, 50, 100, 200 nM), OR sildenafil (50 nM) plus a sarcolemmal (HMR 1098) or a mitochondrial (5-Hydroxydecanoate (5-HD)) K(ATP) channel blocker. Hearts were then subjected to 20 min global, or 35 min regional ischaemia at 37( composite function)C before reperfusion function (aortic output, coronary flow and aortic pressure) and infarct size were documented. Pre-ischaemic, ischaemic and reperfusion myocardial cAMP and cGMP concentrations were determined. RESULTS: Low concentrations of sildenafil (10, 20 and 50 nM) improved reperfusion aortic output (AO) recovery (61.4+/- 4.5%, 64.8 +/- 5.2% and 62.3 +/- 5.0% vs. 45.4 +/- 3.8% for controls (p < 0.05)) and infarct size, while high concentrations (200 nM) worsened AO recovery (24.9 +/- 4.9.0%, p < 0.05). Myocardial cGMP levels of ischaemic tissue were elevated (34.7 +/- 2.4 vs. 27.3 +/- 2.2 pmol/g ww) and cAMP levels were suppressed (0.59 +/- 0.03 vs. 0.87 +/- 0.06 nmol/g ww) in the sildenafil (50 nM) treated hearts. Co-perfusion with sildenafil plus HMR 1098 decreased AO recovery (21.7 +/- 7.6% vs. 62.3 +/- 5.0% for sildenafil alone, p < 0.05) and increased infarct size (29.7 +/- 2.04% vs. 8.6 +/- 2.39% for sildenafil alone, p < 0.05).Similarly, sildenafil plus 5-HD decreased reperfusion AO recovery (44.4 +/- 6.0% vs. 62.3 +/- 5.0% for sildenafil alone, p < 0.05) and increased infarct size (33.8 +/- 1.62% vs. 8.6 +/- 2.39% for sildenafil alone, p < 0.05). CONCLUSIONS: (1) Pretreatment with low concentrations of sildenafil (20-50 nM) improves, while higher concentrations (200 nM) worsen reperfusion function in this model. (2) Low concentrations of sildenafil (20-50 nM) decrease infarct size while the higher concentrations had no effect. (3) These protective properties of low concentrations of sildenafil may be related to its cGMP elevating and cAMP suppressing effects in the ischaemic heart. (4) Possible end-effectors for sildenafil in the ischaemic heart include the mitochondrial and sarcolemmal K(ATP) channel.

The temporal relationship between p38 MAPK and HSP27 activation in ischaemic and pharmacological preconditioning.

An ischaemic preconditioning protocol and subsequent sustained ischaemia were characterized by activation and attenuation of p38 MAPK phosphorylation, respectively. However, the significance of events downstream of p38 MAPK needs investigation. Therefore the temporal relationship between phosphorylation of p38 MAPK and its downstream substrate HSP27 was studied during either an ischaemic or beta-adrenergic preconditioning protocol and during sustained ischaemia. Isolated rat hearts were preconditioned (with or without a p38 MAPK inhibitor, SB203580) with 1 x 5 min or 3 x 5 min global ischaemia or 5 min beta-adrenergic stimulation (10(-7) M isoproterenol), followed by 25 min sustained ischaemia and 30 min reperfusion. Hearts were freeze-clamped at different time intervals and fractionated to determine p38 MAPK and HSP27 phosphorylation, via Western blotting. Significant phosphorylation of cytosolic p38 MAPK and membrane (myo-fibrillar) HSP27 occurred at the end of the first preconditioning episode. However, p38 MAPK phosphorylation disappeared during subsequent preconditioning episodes, while HSP27 phosphorylation was maintained for the duration of the protocol. Similar changes in p38 MAPK and HSP27 occurred with 5 min beta-adrenergic preconditioning. After 25 min ischaemia, significant phosphorylation of cytosolic and membrane HSP27 was observed, while p38 MAPK phosphorylation was attenuated in ischaemic and beta-adrenergic preconditioned compared to non-preconditioned hearts. SB203580-induced abolishment of p38 MAPK and HSP27 phosphorylation during the triggering phase of both preconditioning protocols reversed the changes in these parameters seen after sustained ischaemia. The results suggest that p38 MAPK activation triggers HSP27 phosphorylation during both the preconditioning protocols and during sustained ischaemia. Protection of preconditioned hearts during sustained ischaemia was characterized by phosphorylation of both cytosolic and myofibrillar HSP27.

A mechanism for zinc toxicity in neuroblastoma cells.

Zinc is an important component of proteins essential for normal functioning of the brain. However, it has been shown in vitro that this metal, at elevated levels, can be toxic to cells leading to their death. We investigated possible mechanisms of cell death caused by zinc: firstly, generation of reactive oxygen species, and secondly, the activation of the MAP-kinase pathway. Cell viability was assessed by means of the methyl-thiazolyl tetrazolium salt (MTT) assay and confirmed by tetramethylrhodamine methyl ester (TMRM) staining. We measured the phosphorylation status of Erk and p38 as indicators of MAP-kinase activity, using Western Blot techniques. A time curve was established when neuroblastoma (N2alpha) cells were exposed to 100 microM of zinc for 4, 12, and 24 h. Zinc caused a significant reduction in cell viability as early as 4 h, and indirectly stimulated the accumulation of reactive oxygen species as determined by 2.7 dichlorodihydrofluorescein diacetate (DCDHF) staining and confocal microscopy. Investigation of the MAP-kinase pathway indicated that Erk was downregulated, while p38 was stimulated. Our results therefore led us to conclude that in vitro, zinc toxicity involved the generation of reactive oxygen species and the activation of the MAP-kinase pathway.