Treatment with a fixed dose combination antiretroviral therapy drug containing tenofovir, emtricitabine and efavirenz is associated with cardioprotection in high calorie diet-induced obese rats.

HIV-infection, certain antiretroviral drug classes, especially protease inhibitors (PI), and obesity are associated with increased ischaemic heart disease (IHD) risk. However, the effect of PI-free fixed dose combination (FDC) antiretroviral therapy (ART) on hearts exposed to ischaemia-reperfusion injury (I/R) is unknown, particularly in obesity. This is becoming relevant as World Health Organisation guidelines recommend a FDC ART containing (non-) nucleoside reverse transcriptase inhibitors (tenofovir (TDF), emtricitabine (FTC) and efavirenz (EFV)) as first-line HIV treatment. Additionally, obesity rates are rising in HIV-infected populations, not only in ART-experienced individuals, but also at the time of ART initiation, which may further increase the risk of IHD. Therefore, we investigated the effects of PI-free FDC ART in myocardial I/R-exposed hearts from obese rats. Obesity was induced in male wistar rats via a 16-week high calorie diet. At week 10, treatment with a FDC ART drug containing TDF/FTC/EFV was initiated. Biometric and metabolic parameters, as well as myocardial functional recovery and infract size (IS), and myocardial signalling proteins following I/R were assessed after 16 weeks. Obese rats presented with increased body and intraperitoneal fat mass, elevated triglyceride and TBARS levels, whilst the hearts responded to I/R with impaired functional performance and increased IS. The FDC ART treatment did not alter biometric and metabolic parameters in obese rats. In a novel finding, ART protected obese hearts against I/R as shown by improved functional performance and smaller IS vs. untreated obese hearts. Cardioprotection was underscored by increased myocardial phosphorylated endothelial nitric oxide synthase (eNOS) and reduced AMP-kinase levels. In conclusion, these results demonstrate for the first time, that 6-weeks treatment of obese rats with a FDC ART drug specifically containing TDF/FTC/EFV conferred cardioprotection against I/R. The FDC ART-induced cardioprotection was seemingly unrelated to metabolic changes, but rather due to direct cardiac mechanisms including the up-regulation of myocardial eNOS.

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.

The consequences of long-term glycogen synthase kinase-3 inhibition on normal and insulin resistant rat hearts.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase, discovered as a regulator of glycogen synthase. GSK-3 may regulate the expression of SERCA-2a potentially affecting myocardial contractility. It is known to phosphorylate and inhibit IRS-1, thus disrupting insulin signalling. This study aimed to determine whether myocardial GSK-3 protein and its substrate proteins are dysregulated in obesity and insulin resistance, and whether chronic GSK-3 inhibition can prevent or reverse this. METHODS: Weight matched male Wistar rats were rendered obese by hyperphagia using a special diet (DIO) for 16 weeks and compared to chow fed controls. Half of each group was treated with the GSK-3 inhibitor CHIR118637 (30 mg/kg/day) from week 12 to16 of the diet period. Biometric and biochemical parameters were measured and protein expression determined by Western blotting and specific antibodies. Ca(2+)ATPase activity was determined spectrophotometrically. Cardiomyocytes were prepared by collagenase perfusion and insulin stimulated 2-deoxy-glucose uptake determined. RESULTS: DIO rats were significantly heavier than controls, associated with increased intra-peritoneal fat and insulin resistance. GSK-3 inhibition did not affect weight but improved insulin resistance, also on cellular level. It had no effect on GSK-3 expression but elevated its phospho/total ratio and elevated IRS-2 expression. Obesity lowered SERCA-2a expression and activity while GSK-3 inhibition alleviated this. The phospho/total ratio of phospholamban underscored inhibition of SERCA-2a in obesity. In addition, signs of myocardial hypertrophy were observed in treated control rats. CONCLUSION: GSK-3 inhibition could not reverse all the detrimental effects of obesity but may be harmful in normal rat hearts. It regulates IRS-2, SERCA-2a and phospholamban expression but not IRS-1.

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.

Early cardiovascular changes occurring in diet-induced, obese insulin-resistant rats.

The metabolic syndrome is recognized as a cluster of disturbances associated with obesity, type 2 diabetes and hypertension. Over the past two decades, the number of people with the metabolic syndrome has increased at an alarming rate. This increase is associated with the global epidemic of both obesity and diabetes. Cardiovascular mortality is increased among diabetics and obesity-related insulin-resistant patients, and obesity is currently recognized as independent risk factor for cardiovascular disease. We aimed to establish the effects of a short period of an altered diet on the heart using a rat model of hyperphagia-induced obesity (diet supplemented with sucrose and condensed milk for 8 weeks = DIO) compared to age-matched controls. Isolated, perfused hearts were subjected to global or regional ischaemia/reperfusion. Function on reperfusion was recorded and infarct size determined. A plasma lipid profile was established via HPLC-based methods and proteins involved in metabolic signalling determined either by western blotting or RT-PCR. 8 weeks of diet resulted in whole-body but not myocardial insulin resistance, increased plasma triglyceride and phospholipid levels as well as increased lipid peroxidation. Despite the similar baseline function, hearts from DIO animals showed significantly poorer postischaemic recovery than controls (41.9 % RPP recovery vs 57.9 %, P < 0.05, n = 7-11/group) but surprisingly, smaller infarct size (24.95 ± 1.97 vs 47.26 ± 4.05 % of the area at risk, P < 0.005, n = 8/group). Basal phosphorylation of PKB/Akt was elevated but IRS-1 and SERCA-2 expression severely downregulated. In conclusion, after only 8 weeks of a slight change in diet, the rat heart shows signs of metabolic remodelling. Some of these changes may be protective but others may be detrimental and eventually lead to maladaptation.

PKCε promotes cardiac mitochondrial and metabolic adaptation to chronic hypobaric hypoxia by GSK3ß inhibition.

PKCε is central to cardioprotection. Sub-proteome analysis demonstrated co-localization of activated cardiac PKCε (aPKCε) with metabolic, mitochondrial, and cardioprotective modulators like hypoxia-inducible factor 1α (HIF-1α). aPKCε relocates to the mitochondrion, inactivating glycogen synthase kinase 3ß (GSK3ß) to modulate glycogen metabolism, hypertrophy and HIF-1α. However, there is no established mechanistic link between PKCε, p-GSK3ß and HIF1-α. Here we hypothesized that cardiac-restricted aPKCε improves mitochondrial response to hypobaric hypoxia by altered substrate fuel selection via a GSK3ß/HIF-1α-dependent mechanism. aPKCε and wild-type (WT) mice were exposed to 14 days of hypobaric hypoxia (45 kPa, 11% O(2)) and cardiac metabolism, functional parameters, p-GSK3ß/HIF-1α expression, mitochondrial function and ultrastructure analyzed versus normoxic controls. Mitochondrial ADP-dependent respiration, ATP production and membrane potential were attenuated in hypoxic WT but maintained in hypoxic aPKCε mitochondria (P < 0.005, n = 8). Electron microscopy revealed a hypoxia-associated increase in mitochondrial number with ultrastructural disarray in WT versus aPKCε hearts. Concordantly, left ventricular work was diminished in hypoxic WT but not aPKCε mice (glucose only perfusions). However, addition of palmitate abrogated this (P < 0.05 vs. WT). aPKCε hearts displayed increased glucose utilization at baseline and with hypoxia. In parallel, p-GSK3ß and HIF1-α peptide levels were increased in hypoxic aPKCε hearts versus WT. Our study demonstrates that modest, sustained PKCε activation blunts cardiac pathophysiologic responses usually observed in response to chronic hypoxia. Moreover, we propose that preferential glucose utilization by PKCε hearts is orchestrated by a p-GSK3ß/HIF-1α-mediated mechanism, playing a crucial role to sustain contractile function in response to chronic hypobaric hypoxia.

Kinases and phosphatases in ischaemic preconditioning: a re-evaluation.

Activation of several protein kinases occurs during myocardial ischaemia and during subsequent reperfusion. In contrast to the intensive investigation into the significance of kinase activation in cardioprotection, relatively little is known about the role of the phosphatases in this regard. The aim of this study was to re-evaluate the putative roles of PP1 and PP2A in ischaemia/reperfusion and in triggering ischaemic preconditioning. Isolated perfused working rat hearts were subjected to sustained global (15 or 20 min) or regional ischaemia (35 min), followed by reperfusion. Hearts were preconditioned using global ischaemia (1 x 5 or 3 x 5 min, alternated with 5 min reperfusion). To inhibit both PP1 and PP2A cantharidin (5 muM) was used. To inhibit PP2A only, okadaic acid (7.5 nM) was used. The drugs were administered during the preconditioning protocol, before onset of sustained ischaemia (pretreatment) or during reperfusion. Endpoints were mechanical recovery during reperfusion, infarct size and activation of PKB/Akt, p38 MAPK and ERK p42/p44, as determined by Western blot. Pretreatment of hearts with okadaic acid or cantharidin caused a significant reduction in mechanical recovery after 15 or 20 min global ischaemia. Administration of the drugs during an ischaemic preconditioning protocol abolished functional recovery during reperfusion and significantly increased infarct size. Administration of the drugs during reperfusion had no deleterious effects and increased functional recovery in 3 x PC hearts. To find an explanation for the differential effects of the inhibitors depending on the time of administration, hearts were freeze-clamped at different time points during the perfusion protocol. Administration of cantharidin before 5 min ischaemia activated all kinases. Subsequent reperfusion for 5 min without the drug maintained activation of the kinases until the onset of sustained ischaemia. Cantharidin given during preconditioning was associated with activation of p38MAPK and PKB/Akt during reperfusion after sustained ischaemia. However, administration of the drug during reperfusion only after sustained ischaemia caused activation of both PKB/Akt and ERK p42/p44. Phosphatase inhibition immediately prior to the onset of sustained ischaemia or during preconditioning abolishes protection during reperfusion, while inhibition of these enzymes during reperfusion either had no effect or enhanced the cardioprotective effects of preconditioning. It is proposed that inhibition of phosphatases during reperfusion may prolong the period of RISK activation and hence protect the heart.

Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway.

UNLABELLED: The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. OBJECTIVE: To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. METHODS: Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO(2) incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. RESULTS: (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. CONCLUSIONS: Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.

Melatonin receptor-mediated protection against myocardial ischaemia/reperfusion injury: role of its anti-adrenergic actions.

Melatonin has potent cardioprotective properties. These actions have been attributed to its free radical scavenging and anti-oxidant actions, but may also be receptor mediated. Melatonin also exerts powerful anti-adrenergic actions based on its effects on contractility of isolated papillary muscles. The aims of this study were to determine whether melatonin also has anti-adrenergic effects on the isolated perfused rat heart, to determine the mechanism thereof and to establish whether these actions contribute to protection of the heart during ischaemia/reperfusion. The results showed that melatonin (50 microM) caused a significant reduction in both isoproterenol (10(-7) M) and forskolin (10(-6) M) induced cAMP production and that both these responses were melatonin receptor dependent, since the blocker, luzindole (5 x 10(-6) M) abolished this effect. Nitric oxide (NO), as well as guanylyl cyclase are involved, as L-NAME (50 microM), an NO synthase inhibitor and ODQ (20 microM), a guanylyl cyclase inhibitor, significantly counteracted the effects of melatonin. Protein kinase C (PKC), as indicated by the use of the inhibitor bisindolylmaleimide (50 microM), also play a role in melatonin's anti-adrenergic actions. These actions of melatonin are involved in its cardioprotection: simultaneous administration of L-NAME or ODQ with melatonin, before and after 35 min regional ischaemia, completely abolished its cardioprotection. PKC, on the other hand, had no effect on the melatonin-induced reduction in infarct size. Cardioprotection by melatonin was associated with a significant activation of PKB/Akt and attenuated activation of the pro-apoptotic kinase, p38MAPK during early reperfusion. In summary, the results show that melatonin-induced cardioprotection may be receptor dependent, and that its anti-adrenergic actions, mediated by NOS and guanylyl cyclase activation, are important contributors.

Efficacy of ischaemic preconditioning in the eNOS overexpressed working mouse heart model.

We recently demonstrated that exogenous nitric oxide (NO) acts as a trigger for preconditioning in the isolated rat heart model. There is however little data concerning the effects of elevated cardiac endothelial nitric oxide synthase (eNOS) expression on myocardial tolerance to ischaemia. Similarly, the effects of gender and eNOS overexpression on ischaemic preconditioning is unknown. We hypothesized that: 1) eNOS overexpression increases myocardial tolerance to ischaemia, and, 2) eNOS overexpressed hearts cannot be preconditioned, since the hearts are already maximally protected. Male and female wild-type and transgenic mice that overexpress eNOS exclusively in cardiac myocytes were perfused in the working heart mode with a modified Krebs-Henseleit buffer at a pre-load of 12.5 mm Hg and afterload of 50 mm Hg. Cardiac output, coronary flow, peak aortic systolic pressure and total work were determined before hearts were preconditioned by 4x5 min cycles of ischaemia/reperfusion, and then subjected to 20 min total global ischaemia, followed by reperfusion. Reperfusion function and myocardial infarct size were used as endpoints. Pre-ischaemic mechanical function (rate pressure product and cardiac output) was similar for wild-type and transgenic mice of both sexes. The eNOS overexpressed hearts had smaller infarcts than the hearts from their wild-type littermates (26.9+/-1.4% vs. 37.0+/-2.1% for controls, P<0.05). Preconditioning the eNOS overexpressed hearts resulted in infarct sizes comparable with control non-preconditioned hearts (27.5+/-2.0% vs. 26.9+/-1.4% for controls). Myocardial cGMP levels were elevated during sustained ischaemia in the transgenic hearts when compared with wild-type hearts (22.43+/-1.63 pmol/g ww vs 16.54+/-1.48 pmol/g ww, P<0.05). Preconditioning also elevated myocardial cGMP levels during sustained ischaemia in the wild-type hearts (26.77+/-2.81 pmol/g ww, P<0.05). We conclude that: 1) basal mechanical function is similar for both wild-type and transgenic mice of both sexes, 2) reperfusion function and infarct size was also similar for both sexes under both control conditions and after preconditioning, 3) the transgenic mice are more tolerant of ischaemia as reflected by their smaller myocardial infarcts, and, 4) the eNOS overexpressed mouse heart cannot be preconditioned regardless of whether mechanical function or infarct size is used as an end-point. These hearts may be maximally protected against ischaemia/reperfusion injury by their elevated endogenous NO levels.

p38-MAPK and PKB/Akt, possible role players in red palm oil-induced protection of the isolated perfused rat heart?

It has been shown that dietary red palm oil (RPO) supplementation improves reperfusion function. However, no exact protective cellular mechanisms have been established. To determine a potential mechanism for functional improvement, we examined the regulation of both mitogen-activated protein kinases (MAPKs) and PKB/Akt in the presence and absence of dietary RPO supplementation in ischemia/reperfusion-induced injury. Wistar rats were fed a control diet or control diet plus 7 g RPO/kg diet for 6 weeks. Hearts were excised and mounted on an isolated working heart perfusion apparatus. Cardiac function was measured before and after hearts were subjected to 25 min of total global ischemia. Hearts subjected to the same conditions were freeze clamped and used to characterize the degree of phosphorylation of extracellular signal-regulated kinase, p38, c-Jun NH(2)-terminal protein kinase (JNK) and PKB/Akt. Dietary RPO supplementation significantly improved aortic output recovery (72.1 +/- 3.2% vs. 54.0 +/- 3.2%, P < .05). This improved aortic output recovery was associated with significant increases in p38 and PKB/Akt phosphorylation during reperfusion when compared with control hearts. Furthermore, a significant decrease in JNK phosphorylation and attenuation of poly(ADP-ribose) polymerase cleavage occurred in the RPO-supplemented group during reperfusion. Our results suggest that dietary RPO supplementation caused differential phosphorylation of the MAPKs and PKB/Akt during ischemia/reperfusion-induced injury. These changes in phosphorylation were associated with improved functional recovery and reduced cleavage of an apoptotic marker, arguing that dietary RPO supplementation may confer protection via the MAPK and PKB/Akt signaling pathways during ischemia/reperfusion-induced injury.

H-89, a non-specific inhibitor of protein kinase A, promotes post-ischemic cardiac contractile recovery and reduces infarct size.

Myocardial ischemia is associated with increased production of cyclic adenosine monophosphate (cAMP), with potentially deleterious effects. We hypothesized that the ischemia-induced activation of cAMP-dependent protein kinase A (PKA), could beneficially be inhibited by a PKA-inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline-sulfonamide (H-89). H-89 when given to isolated perfused rat hearts before 30 minutes of global ischemia-reperfusion improved postischemic function and decreased infarct size. In another series, H-89 administered prior to preconditioning by 10 minutes of transient global ischemia decreased PKA activity (measured at the end of the preconditioning protocol) and augmented postischemic mechanical recovery. H-89 given for 5 minutes before the 10 minutes of transient ischemia further decreased infarct size from 13.4 +/- 1.0% (preconditioning alone) to 7.0 +/- 0.93 (P < 0.01). In a third series, forskolin (0.3 muM, 5 minutes, 10 minutes washout prior to ischemia) increased PKA activity and reduced infarct size. Prior H-89 decreased PKA activity after 5 minutes of forskolin and further reduced infarct size versus forskolin alone. In conclusion, three procedures increased postischemic recovery and reduced infarct size: H-89; preconditioning by transient ischemia; or forskolin as a preconditioning-mimetic. PKA-inhibition by H-89 further decreased infarct size beyond preconditioning or forskolin. Despite the reservation that H-89 could be non-selective in its actions, we propose H-89 as a candidate cardioprotective agent.

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.

Nitric Oxide synthase (NOS) does not contribute to simulated ischaemic preconditioning in an isolated rat cardiomyocyte model.

UNLABELLED: It is widely accepted that nitric oxide (NO) is a trigger and mediator of late ischaemic preconditioning (IP), however its role in classic (protection observed within 2-4 hours after the IP stimulus) IP is less certain. In addition, the contribution of cardiomyocyte nitric oxide synthase (NOS) activation to NO production in ischaemia is unknown. The aim of this study was therefore to investigate the role of NOS, NO, reactive oxygen species (ROS) and cGMP in IP in an isolated cardiomyocyte model. METHODS: Adult rat cardiomyocytes were isolated by collagenase perfusion. Hypoxia was induced by covering pelleted cardiomyocytes with mineral oil. The IP protocol was one 10 min hypoxia/20 min reoxygenation cycle, followed by 2 hr sustained hypoxia. Non-IP cells were subjected to 2 hr sustained hypoxia only. The contribution of NO was investigated by NOS inhibition (L-NAME 50 microM) or by pre-treatment of cells with a NO donor (SNP 100 microM), and that of ROS by inclusion of ROS scavengers (MPG and N-acetyl-cysteine) or pre-treatment with H(2)O(2). End-points were cellular cGMP content and cell viability as assessed by trypan blue exclusion (TBE) and cell morphology. RESULTS: IP significantly improved myocyte viability (54% increase in TBE) at the end of sustained hypoxia. Treatment of cells with L-NAME and ROS scavengers during either the IP protocol or during sustained hypoxia had no effect on cell viability after 2 hr hypoxia, whereas viability of non-IP cells treated with L-NAME during sustained hypoxia improved significantly. cGMP levels were reduced in IP cells. Pre-treatment with SNP and H(2)O(2) did not mimic IP. CONCLUSIONS: IP conferred cardioprotection in isolated cardiomyocytes. Protection in this model was not due to activation of cardiomyocyte NOS or ROS production. However, NOS activation induced by sustained hypoxia, appeared to be harmful to non-IP cells.

Manipulation of papillary muscle cyclic nucleotides during anoxia-reoxygenation: effects on contractility.

Endogenous nitric oxide and the accompanying cGMP formation has been postulated to play a role in the pathophysiology of myocardial anoxia-reoxygenation. A direct relationship between cGMP and the alterations observed in contractility under these conditions has never been demonstrated. In this study, cGMP in rat papillary muscles during anoxia and reoxygenation was correlated with mechanical function. Isolated papillary muscles were stimulated continuously and made anoxic for 40 min after a 2-hour stabilisation period. Anoxia caused an abbreviated contraction curve by decreasing the maximum contraction strength, time to peak contraction and relaxation time, accompanied by a significant decrease in tissue cGMP and cAMP levels (controls: 29.09 +/- 1.62 and 568.8 +/- 35.65; anoxia:16.62 +/- 1.51 and 403.3 +/- 30.19 pmoles/gww), which partially returned to pre-anoxic values upon reoxygenation. cGMp levels were significantly elevated by addition of 8-Br-cGMP (a cGMP analogue), but this elevation (154.4 +/- 20.89 pmoles/gww), had no effects on the contractility pattern of muscles during normoxia, anoxia or reoxygenation, suggesting that in isolated ventricular muscle, cGMP levels play a minor role in regulating muscle contractility.