Erna Marais

Nitric oxide triggers classic ischemic preconditioning.

Abstract: The role of NO in the classic ischemic preconditioning phenomenon of the myocardium is not well defined, and was investigated by using the isolated perfused rat heart as a model. Hearts were preconditioned with 3 × 5 minute ischemia in the presence and absence of the NOS inhibitors l‐NAME (50 μM) and l‐NNA (50 μM), and the guanylyl cyclase inhibitor ODQ (20 μM). These inhibitors significantly attenuated the protective effect of preconditioning against 25‐min global ischemia (as measured by functional recovery), specifically if administered during the triggering phase. Cyclic infusions (3 × 5 min) of the NO‐donors SNAP (50 μM) and SNP (100 μM) elicited protection against both 25‐min global or low‐flow ischemia. Hearts preconditioned with NO donors displayed significantly superior functional reserve, if stimulated with adrenaline, compared to hearts preconditioned with ischemia. Although the NO donors SNAP and SNP both activated p38 MAPK during the preconditioning protocol, protection was accompanied by significantly decreased p38 MAPK activity during sustained ischemia, as was the case in ischemic preconditioning. We conclude that (1) NO is a trigger for classic preconditioning, (2) cGMP generation plays an important role in its protection, (3) attenuation of p38 MAPK during sustained ischemia accompanies NO preconditioning and may mediate cardiac protection, and (4) preconditioning with NO may be more advantageous than using ischemia.

Mitochondrial catastrophe during doxorubicin‐induced cardiotoxicity: a review of the protective role of melatonin

Anthracyclines, such as doxorubicin, are among the most valuable treatments for various cancers, but their clinical use is limited due to detrimental side effects such as cardiotoxicity. Doxorubicin‐induced cardiotoxicity is emerging as a critical issue among cancer survivors and is an area of much significance to the field of cardio‐oncology. Abnormalities in mitochondrial functions such as defects in the respiratory chain, decreased adenosine triphosphate production, mitochondrial DNA damage, modulation of mitochondrial sirtuin activity and free radical formation have all been suggested as the primary causative factors in the pathogenesis of doxorubicin‐induced cardiotoxicity. Melatonin is a potent antioxidant, is nontoxic, and has been shown to influence mitochondrial homeostasis and function. Although a number of studies support the mitochondrial protective role of melatonin, the exact mechanisms by which melatonin confers mitochondrial protection in the context of doxorubicin‐induced cardiotoxicity remain to be elucidated. This review focuses on the role of melatonin on doxorubicin‐induced bioenergetic failure, free radical generation, and cell death. A further aim is to highlight other mitochondrial parameters such as mitophagy, autophagy, mitochondrial fission and fusion, and mitochondrial sirtuin activity, which lack evidence to support the role of melatonin in the context of cardiotoxicity.

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.

Comparison Between Ischaemic and Anisomycin-Induced Preconditioning: Role of p38 MAPK

To further evaluate the significance of p38 MAPK as trigger or mediator in ischaemic preconditioning, anisomycin and SB 203580 were used to manipulate its activation status. Special attention was given to the concentration of the drugs and protocols used.The isolated perfused rat heart, subjected to either 25 min global ischaemia or 35 min regional ischaemia, was used as experimental model. This was preceded by anisomycin (2 or 5 μM: 3 × 5 min; 5 μM: 5 min or 10 min; 5 μM: 10 min + 10 min washout or 20 μM: 20 min) or SB 203580 (2 μM: 3 × 5 min; before and during 3 × 5 min or 1 × 5 min ischaemic preconditioning; 10 min). Endpoints were functional recovery during reperfusion and infarct size.Anisomycin, regardless of the protocol, reduced infarct size, but did not improve functional recovery. In a number of experiments activation of JNK by anisomycin was blocked by SP 600125 (10 μM). SP 600125 had no effect on the anisomycin-induced reduction in infarct size. SB 203580 when administered for 10 min before sustained ischaemia, improved functional recovery and reduced infarct size. SB 203580 could not abolish the beneficial effects of a multi-cycle preconditioning protocol, but it significantly reduced the outcome of 1 × 5 min preconditioning. In all hearts improved functional recovery and reduction in infarct size were associated with attenuation of p38 MAPK activation during sustained ischaemia-reperfusion.The results indicate that activation of p38 MAPK acts as a trigger of preconditioning, while attenuation of its activation is a prerequisite for improved recovery and a reduction in infarct size.

Serial changes in the myocardial β-adrenergic signalling system in two models of non-insulin dependent diabetes mellitus

Since it was reported in 1991 by Schaffer et al. that myocardial contractile responsiveness was altered in NIDDM in the absence of alterations in the β-adrenergic receptor population, researchers have been seeking a post-receptor defect to account for this. The present study addresses this issue by comparing alterations occurring in the myocardial β-receptor signalling pathway in two different models of rat NIDDM, as well as the response of the pathway after stimulation with isoproterenol in the presence or absence of insulin. The characteristics of the β-receptor population, adenylyl cyclase activity and cAMP levels were determined at three different ages. The main results demonstrate that: (i) the two models of NIDDM myocardium differ biochemically; (ii) the β-adrenergic signalling system of the insulin deficient model was altered more than the hyperinsulinemic model and (iii) the observed exaggerated cAMP response of NIDDM hearts after stimulation with a β-adrenergic agonist is in contrast with lower responsivity.

The differential effects of FTY720 on functional recovery and infarct size following myocardial ischaemia/reperfusion : cardiovascular topics

Summary Aim The aim of this study was to evaluate the effects of the sphingosine analogue, FTY720 (Fingolimod), on the outcomes of myocardial ischaemia/reperfusion (I/R) injury. Methods Two concentrations of FTY720 (1 or 2.5 μM) were administered either prior to (PreFTY), or following (PostFTY) 20 minutes’ global (GI) or 35 minutes’ regional ischaemia (RI) in the isolated, perfused, working rat heart. Functional recovery during reperfusion was assessed following both models of ischaemia, while infarct size (IFS) was determined following RI. Results FTY720 at 1 μM exerted no effect on functional recovery, while 2.5 μM significantly impaired aortic output (AO) recovery when administered prior to GI (% recovery: control: 33.88 ± 6.12% vs PreFTY: 0%, n = 6–10; p < 0.001), as well as before and after RI (% recovery: control: 27.86 ± 13.22% vs PreFTY: 0.62%; p < 0.05; and PostFTY: 2.08%; p = 0.0585, n = 6). FTY720 at 1 μM administered during reperfusion reduced IFS [% of area at risk (AAR): control: 39.89 ± 3.93% vs PostFTY: 26.56 ± 4.32%, n = 6–8; p < 0.05), while 2.5 μM FTY720 reduced IFS irrespective of the time of administration (% of AAR: control: 39.89 ± 3.93% vs PreFTY: 29.97 ± 1.03%; and PostFTY: 30.45 ± 2.16%, n = 6; p < 0.05). Conclusion FTY720 exerted divergent outcomes on function and tissue survival depending on the concentration administered, as well as the timing of administration.

Melatonin and cardioprotection against ischaemia/reperfusion injury: What's new? A review

Melatonin is a pleiotropic hormone with several functions. It binds to specific receptors and to a number of cytosolic proteins, activating a vast array of signalling pathways. Its potential to protect the heart against ischaemia/reperfusion damage has attracted much attention, particularly in view of its possible clinical applications. This review will focus mainly on the possible signalling pathways involved in melatonin‐induced cardioprotection. In particular, the role of the melatonin receptors and events downstream of receptor activation, for example, the reperfusion injury salvage kinase (RISK), survivor activating factor enhancement (SAFE) and Notch pathways, the sirtuins, nuclear factor E2–related factor 2 (Nrf2) and translocases in the outer membrane (TOM70) will be discussed. Particular attention is given to the role of the mitochondrion in melatonin‐induced cardioprotection. In addition, a brief overview will be given regarding the status quo of the clinical application of melatonin in humans.

Melatonin improves cardiac and mitochondrial function during doxorubicin-induced cardiotoxicity: A possible role for peroxisome proliferator-activated receptor gamma coactivator 1-alpha and sirtuin activity?

ABSTRACT Mitochondrial dysfunction is a central element in the development of doxorubicin (DXR)‐induced cardiotoxicity. In this context, melatonin is known to influence mitochondrial homeostasis and function. This study aimed to investigate the effects of melatonin on cardiac function, tumor growth, mitochondrial fission and fusion, PGC1‐&agr; and sirtuin activity in an acute model of DXR‐induced cardiotoxicity. During the in vitro study, H9c2 rat cardiomyoblasts were pre‐treated with melatonin (10 &mgr;M, 24 h) followed by DXR exposure (3 &mgr;M, 24 h). Following treatment, cellular ATP levels and mitochondrial morphology were assessed. In the in vivo study, female Sprague Dawley rats (16 weeks old), were inoculated with a LA7 rat mammary tumor cell line and tumors were measure daily. Animals were injected with DXR (3 × 4 mg/kg) and/or received melatonin (6 mg/kg) for 14 days in their drinking water. Rat hearts were used to conduct isolated heart perfusions to assess cardiac function and thereafter, heart tissue was used for immunoblot analysis. DXR treatment increased cell death and mitochondrial fission which were reduced with melatonin treatment. Cardiac output increased in rats treated with DXR + melatonin compared to DXR‐treated rats. Tumor volumes was significantly reduced in DXR + melatonin‐treated rats on Day 8 in comparison to DXR‐treated rats. Furthermore, DXR + melatonin treatment increased cellular ATP levels, PGC1‐&agr; and SIRT1 expression which was attenuated by DXR treatment. These results indicate that melatonin treatment confers a dual cardio‐protective and oncostatic effect by improving mitochondrial function and cardiac function whilst simultaneously retarding tumor growth during DXR‐induced cardiotoxicity. Graphical abstract Figure. No Caption available. HighlightsMelatonin is cardio‐protective and oncostatic during DXR‐induced cardiotoxicity.DXR + melatonin treatment increases ATP, PGC1‐&agr; and SIRT1expression attenuated by DXR.Melatonin enhances efficacy of DXR treatment by reducing breast tumor volume.Melatonin improves cardiac function and reduces apoptosis during DXR treatment.

The Role of the Beta-Adrenergic Signal Transduction Pathway in Myocardial Protection

Beta-adrenergic activation is a major factor causing myocardial damage in the context of ischemia. Activation of the beta-adrenergic signal transduction pathway can, however, also elicit protective responses in the myocardium. Activation of the beta-adrenergic signal transduction pathway participates in the protective effect of ischemic preconditioning, and administration of catecholaminergic agents such as isoproterenol and noradrenaline can elicit pharmacological preconditioning. Protection induced by beta-adrenergic activation (beta-adrenergic preconditioning) elicits both classic (early) and delayed (late) preconditioning and utilizes adenosine to mediate acute protection, while utilizing NO in pharmacological late beta-preconditioning. It is unclear which beta-adrenergic receptor subtype is involved in mediating protection, with evidence for a role of both the “harmful” receptor, beta1, and the beta2 adrenergic receptor. Our own findings suggest cAMP and PKA as the second messengers involved in this ability of beta-adrenergic activation to activate a protective response during the triggering phase of protection, whereas attenuation of activation of p38 MAPK during ischemia is involved in protection against ischemia-mediated necrosis and apoptosis.

Ischaemic and Pharmacological Preconditioning Is Associated with Attenuation of p38 MAPK Activation During Sustained Ischaemia and Reperfusion

The stress kinase, p38 mitogen activated protein kinase (p38 MAPK), is known to be activated by both ischaemia and reperfusion. Whether this activation is beneficial or deleterious is still a matter of debate. The aims of this study were to (i) evaluate the activation pattern of p38 MAPK during a multi-cycle preconditioning protocol, during sustained (index) ischaemia and during reperfusion (ii) use transient β-adrenergic stimulation to mimic ischaemic preconditioning and to study its effects on p38 MAPK activation and (iii) reevaluate the effects of p38 MAPK blockade by SB 203580 on cardioprotection elicited by ischaemic preconditioning as well as by β-adrenergic preconditioning. The isolated perfused working rat heart was subjected to a preconditioning protocol of 3 X 5 min global ischaemia or 5 min isoproterenol (10-7M) followed by 5 min washout. All hearts were then subjected to 25 min global ischaemia and 30 min reperfusion during which time mechanical function was monitored. Hearts were freeze-clamped at different time intervals for subsequent analysis of p38 MAPK activation using Western blotting and a p38 MAPK phospho-antibody (Thr 180/Tyr 182).