Johan A. Moolman

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.

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.

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.

Do binucleate cardiomyocytes have a role in myocardial repair? Insights using isolated rodent myocytes and cell culture

Neonatal and adult cardiomyocytes were isolated from rat hearts. Some of the adult myocytes were cultured to allow for cell dedifferentiation, a phenomenon thought to mimic cell changes that occur in stressed myocardium, with myocytes regressing to a fetal pattern of metabolism and stellate neonatal shape. Using fluorescence deconvolution microscopy, cells were probed with fluorescent markers and scanned for a number of proteins associated with ion control, calcium movements and cardiac function. Image analysis of deconvoluted image stacks and sequential real-time image recordings of calcium transients of cells were made. All three myocyte groups were predominantly comprised of binucleate cells. Clustering of proteins to a single nucleus was a common observation, suggesting that one nucleus is active in protein synthesis pathways, while the other nucleus assumes a ‘dormant’ or different role and that cardiomyocytes might be mitotically active even in late development, or specific protein syntheses could be targeted and regulated for reintroduction into the cell cycle. Such possibilities would extend cardiac disease associated stem cell research and therapy options, while producing valuable insights into developmental and death pathways of binucleate cardiomyocytes (word count 183).

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).