Sonia Genade

Ischemic Preconditioning and the β-Adrenergic Signal Transduction Pathway

Background—Previous studies from our laboratory showed cyclic increases in tissue cAMP during a multiple-cycle preconditioning (PC) protocol, followed by attenuated cAMP accumulation during sustained ischemia. The aim of this study was to determine whether ischemia-induced activation of the β-adrenergic signaling pathway could act as a trigger in eliciting protection. Methods and Results—Isolated perfused rat hearts were preconditioned by 3×5 minutes of global ischemia, interspersed by 5 minutes of reperfusion. β-Adrenergic responsivity was assessed by measurement of tissue cAMP generation after β-adrenergic agonist administration at the end of the PC protocol. Tissue cAMP, adenylyl cyclase, and protein kinase A (PKA) activities and β-adrenergic receptor characteristics were assessed at different times. The role of cAMP generation in eliciting PC was studied by investigation of functional recovery during reperfusion after 25 minutes of global ischemia after (1) cAMP increases in the trigger period were pr...

Short- and long-term effects of melatonin on myocardial post-ischemic recovery

Abstract:  Melatonin, the chief secretory product of the pineal gland, has been shown to protect the heart against ischemia–reperfusion injury. This was attributed to its free radical scavenging and broad‐spectrum antioxidant properties. The possibility that melatonin may act via its receptor and intracellular signaling, has not yet been addressed in this regard. In all previous studies, only the acute effects of melatonin on the heart, were evaluated. The aims of the present study were to: (i) compare the acute and long‐term effects of melatonin on infarct size and functional recovery of the ischemic heart, and (ii) evaluate the role of the melatonin receptor in cardioprotection. For evaluation of the short‐term effects of melatonin on contractile recovery and infarct size, the isolated perfused working rat heart was subjected to 20 min global ischemia or 35 min regional ischemia respectively, and melatonin (25–50 μm) administered either before and during reperfusion, or before ischemia or during reperfusion after ischemia. The melatonin receptor was manipulated using luzindole and N‐acetyltryptamine. The long‐term effects of melatonin were evaluated 24 hr after melatonin administration (2.5 or 5.0 mg/kg, i.p.) or after oral administration for 7 days (20 or 40 μg/mL). Infarct size and mechanical recovery during reperfusion of the working heart were used as endpoints. Melatonin (50 μm), when administered either before and during reperfusion after ischemia or during reperfusion only, significantly improved cardiac output and work performance and reduced infarct size compared with untreated controls. Luzindole (5 μm), a melatonin receptor antagonist, abolished these cardioprotective effects. Long‐term administration of melatonin (i.p. or orally for 7 days) caused a significant reduction in infarct size of hearts subjected to 35 min regional ischemia. The cardioprotection persisted for 2–4 days after discontinuation of treatment. In summary, the results obtained suggest that melatonin induces short‐ as well as long‐term protection and that the melatonin receptor is also involved in its cardioprotective actions.

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

Abstract:  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 μm) 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 × 10−6 m) abolished this effect. Nitric oxide (NO), as well as guanylyl cyclase are involved, as l‐NAME (50 μm), an NO synthase inhibitor and ODQ (20 μm), a guanylyl cyclase inhibitor, significantly counteracted the effects of melatonin. Protein kinase C (PKC), as indicated by the use of the inhibitor bisindolylmaleimide (50 μm), 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.

Halothane protects the isolated rat myocardium against excessive total intracellular calcium and structural damage during ischemia and reperfusion

A recent study from our laboratory demonstrated halothane to be a powerful protectant of the isolated rat heart during reperfusion after normothermic cardioplegic arrest. It was speculated that this protective effect might be due to prevention of excessive intracellular calcium. The aim of the present study was to evaluate the effect of halothane on the total intracellular calcium (Ca2+) content and on myocardial structure both at the end of normothermic cardioplegic arrest and at the end of reperfusion. Isolated perfused rat hearts were perfused for a control period of 30 min, followed by 40 min of normothermic cardioplegic arrest with or without reperfusion for 30 min. Halothane (1.5%) was administered continuously before and after arrest. Halothane caused a significant decrease of intracellular Ca2+ at the end of normothermic cardioplegic arrest and after reperfusion. Myocardial morphology was assessed by extensive light microscopy and ultrastructure was evaluated by electron microscopy. Grading of ischemic damage showed that exposure to normothermic cardioplegia resulted in marked ischemic injury, regardless of whether the hearts were treated with halothane. Reperfusion in the presence of halothane caused a significant reversal of ischemic damage and almost complete ultrastructural repair, whereas untreated hearts still exhibited severe edema, contracture, and contracture bands. Our results indicate that the beneficial effects of halothane on myocardial structural recovery during reperfusion is associated with a reduction in excessive intracellular Ca2+. The exact mechanism of this protective action is under investigation.

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

AbstractAn 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 β–adrenergic preconditioning protocol and during sustained ischaemia.Isolated rat hearts were preconditioned (with or without a p38 MAPK inhibitor, SB203580) with 1 × 5 min or 3 × 5 min global ischaemia or 5 min β–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 β–adrenergic preconditioning. After 25 min ischaemia, significant phosphorylation of cytosolic and membrane HSP27 was observed, while p38 MAPK phosphorylation was attenuated in ischaemic and β–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.

Role of cyclic nucleotide phosphodiesterases in ischemic preconditioning

Several signal transduction pathways have been implicated in the mechanism of protection induced by ischemic preconditioning (PC). For example, stimulation of a variety of G-protein coupled receptors results in stimulation of protein kinase C (PKC) which has been suggested to act as common denominator in eliciting protection. PC also significantly attenuated cAMP accumulation during sustained ischemia, suggesting involvement of an anti-adrenergic mechanism. The aim of this study was to evaluate the β-adrenergic signal transduction pathway (as evidenced by changes in tissue cAMP and cAMP- and cGMP-phosphodiesterases during the PC protocol as well as during sustained ischemia. Isolated perfused rat hearts were preconditioned by 3 × 5 min global ischemia (PC1,2,3) interspersed by 5 min reperfusion, followed by 25 min global ischemia. Tissue cAMP- and cGMP-PDE activity as well as cAMP and cGMP levels were determined at different time intervals during the PC protocol and sustained ischemia. Tissue cAMP increased with each PC ischemic event and normalized upon reperfusion, while PDE activity showed the opposite, viz a reduction during ischemia and an increase during reperfusion. Except for PC1, tissue cGMP showed similar fluctuations. Throughout 25 min sustained ischemia, cAMP- and cGMP-PDE activities were higher in PC than in nonpreconditioned hearts, associated with a significantly lesser accumulation in cAMP and higher cGMP levels in the former. Fluctuations in cyclic nucleotides during preconditioning were associated with concomitant changes in PDE activity, while the attenuated β-adrenergic response of preconditioned hearts during sustained ischemia may partially be due to increased PDE activity.

No evidence for mediation of ischemic preconditioning by alpha1-adrenergic signal transduction pathway or protein kinase C in the isolated rat heart

SummaryThe purpose of this study was to elucidate the role of activation of the alpha1-adrenergic signal transduction pathway and of protein kinase C (PKC) in the mechanism of protection of functional recovery by ischemic preconditioning in the isolated perfused rat heart. After a stabilization period, nonpreconditioned and preconditioned isolated perfused rat hearts were subjected to sustained ischemia for 25 and 30 minutes of reperfusion. Preconditioning consisted of three episodes of 5 minutes of ischemia, interspersed with 5 minutes of reperfusion. The endpoint was postischemic functional recovery. The effectiveness of preconditioning in the presence of the alpha1-adrenergic blocker prazosin, the selective PKC blockers chelerythrine and bisindolylmaleimide (BIM), and the ability of repetitive alpha1-adrenergic activation to mimic preconditioning were compared with the appropriate nonpreconditioned and preconditioned control groups. Alpha1-adrenergic blockade with prazosin (3×10-7 M) during the preconditioning phase did not abolish the protective effect of preconditioning on functional recovery, and repeated intermittent alpha1-adrenergic activation with phenylephrine in different concentrations (1×10-8 to 3× 10-5 M) did not mimic the protective effect of preconditioning. PKC blockade with the selective PKC inhibitors, chelerythrine (10 μM) and BIM (4 μM), did not abolish the protective effect of preconditioning on functional recovery is isolated perfused rat hearts when given either during the preconditioning phase or shortly before the onset of sustained ischemia. The characteristic metabolic changes of preconditioning during sustained ischemia, namely, energy sparing as manifested in reduced accumulation of lactate, were also not abolished by preconditioning in the presence of selective PKC blockers. We conclude that no evidence could be found for alpha1-adrenergic or PKC activation in the mechanism of ischemic preconditioning in the isolated rat heart.

Halothane does have protective properties in the isolated ischemic rat heart

To determine whether halothane has protective effects on the ischemic heart, the influence of various concentrations (0.5%-1.5%) of halothane on metabolic and functional recovery during reperfusion after 60-min hypothermic (20 degrees C) and 40-min normothermic cardioplegic arrest was determined in the isolated rat heart. Halothane was administered either before and after arrest or intermittently during arrest. Hearts not receiving halothane demonstrated a reduction in adenosine triphosphate (ATP) content from a control value of 20.35 +/- 1.66 mumol/g dry wt (mean +/- SEM) (before arrest) to 9.34 +/- 1.12 mumol/g dry wt at the end of arrest (P less than 0.001). The myocardial ATP content, when measured 20 min after arrest and during reperfusion, remained decreased (9.57 +/- 0.62 mumol/g dry wt). Under these experimental conditions, aortic flow was reduced from 43.62 +/- 2.40 mL/min before arrest to 1.80 +/- 1.80 mL/min 20 min after arrest and during reperfusion (P less than 0.001). The administration of adrenaline after 20 min of reperfusion resulted in partial recovery to 22.01 +/- 8.36 mL/min. Administration of halothane (0.5%) before the cardioplegic period was associated with a reduction of ATP at the end of the normothermic arrest (4.02 +/- 0.38 mumol/g dry wt; P less than 0.01), but the ATP increased significantly (13.45 +/- 0.32 mumol/g dry wt) when measured after the arrest and after 20 min of reperfusion and stimulation with adrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

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 μM, 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.

Enflurane and isoflurane reduce reperfusion dysfunction in the isolated rat heart.

We evaluated the possible cardioprotective effects of enflurane (E) and isoflurane (I) in isolated rat hearts subjected to 40 min normothermic arrest. After reperfusion, hearts were stimulated with adrenaline to evaluate their systolic reserves. In hearts not receiving I or E, adenosine triphosphate (ATP) was reduced from 23.0 ± 0.8 to 9.3 ± 1.1 μmol/g dry weight (means ± SEM; P < 0.001) after arrest. This was associated with a significant reduction in ventricular work (Wt) from 13.6 ± 0.7 to 1.6 ± 0.7 mW (P < 0.001). Adrenaline partially restored Wt but not the ATP. E and I given only during normothermic arrest (in the cardioplegic solution) resulted in reductions in ATP similar to the hearts not receiving the drugs. However, on reperfusion and subsequent administration of adrenaline, hearts subjected to the anesthetic drugs performed as well as hearts before arrest. For example, in hearts not exposed to I or E, the Wt after the elective arrest was 1.55 ± 0.05% (mean ± SEM) of the pre-arrest value. This was significantly less than hearts exposed to either one of the inhalational agents (40.02 ± 3.49% of the pre-arrest value; P < 0.0001). Adrenaline improved function in hearts which did not receive I or E to 55.02 ± 12.80% of the pre-arrest value, but this was significantly less than the Wt performed by the hearts exposed to the anesthetic agents (122.67 ± 7.78% of pre-arrest value; P < 0.001). This beneficial effect of I and E during reperfusion probably is mediated by the effect of the anesthetic agents on Ca2+ slow channels. The effect could not be ascribed to depression of global myocardial contractile function associated with I and E.