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1.  Dual role of nNOS in ischemic injury and preconditioning 
BMC Physiology  2010;10:15.
Nitric oxide (NO) is cardioprotective and a mediator of ischemic preconditioning (IP). Endothelial nitric oxide synthase (eNOS) is protective against myocardial ischemic injury and a component of IP but the role and location of neuronal nitric oxide synthase (nNOS) remains unclear. Therefore, the aims of these studies were to: (i) investigate the role of nNOS in ischemia/reoxygenation-induced injury and IP, (ii) determine whether its effect is species-dependent, and (iii) elucidate the relationship of nNOS with mitoKATP channels and p38MAPK, two key components of IP transduction pathway.
Ventricular myocardial slices from rats and wild and nNOS knockout mice, and right atrial myocardial slices from human were subjected to 90 min ischemia and 120 min reoxygenation (37°C). Specimens were randomized to receive various treatments (n = 6/group). Both the provision of exogenous NO and the inhibition of endogenous NO production significantly reduced tissue injury (creatine kinase release, cell necrosis and apoptosis), an effect that was species-independent. The cardioprotection seen with nNOS inhibition was as potent as that of IP, however, in nNOS knockout mice the cardioprotective effect of non-selective NOS (L-NAME) and selective nNOS inhibition and also that of IP was blocked while the benefit of exogenous NO remained intact. Additional studies revealed that the cardioprotection afforded by exogenous NO and by inhibition of nNOS were unaffected by the mitoKATP channel blocker 5-HD, although it was abrogated by p38MAPK blocker SB203580.
nNOS plays a dual role in ischemia/reoxygenation in that its presence is necessary to afford cardioprotection by IP and its inhibition reduces myocardial ischemic injury. The role of nNOS is species-independent and exerted downstream of the mitoKATP channels and upstream of p38MAPK.
PMCID: PMC2927582  PMID: 20707900
2.  Effect of the degree of ischaemic injury and reoxygenation time on the type of myocardial cell death in man: role of caspases 
BMC Physiology  2005;5:14.
The importance of apoptosis in the injury sustained by the human myocardium during ischaemia and reoxygenation and the underlying mechanisms remain unclear. To quantify apoptosis and necrosis induced by simulated ischaemia/reoxygenation in the human atrial myocardium, free-hand sections of right atrial appendage (n = 8/group) were subjected to 90 minutes simulated ischaemia followed by 2, 8 and 24 hours reoxygenation.
Apoptosis, as assessed by TUNEL, was greater than necrosis after 90 minutes simulated ischaemia and 2 hours reoxygenation (35.32 ± 3.22% vs 13.55 ± 1.3%; p < 0.05) but necrosis was greater than apoptosis by 24 hours reoxygenation (45.20 ± 2.75% vs 4.82 ± 0.79%; p < 0.05). Total caspase activation was similar after 90 minutes simulated ischaemia followed by 2 hours and 24 hours reoxygenation (515270 ± 99570 U vs 542940 ± 95216 U; p = NS). However, caspase-3 like activation was higher at 2 hours than at 24 hours reoxygenation (135900 ± 42200 U vs 54970 ± 19100 U; p < 0.05). Inhibition of caspase-3 by z.DEVD.fmk (70 nM) almost completely abolished apoptosis from 23.26 ± 2.854% to 0.73 ± 0.28 % (p < 0.05), without affecting necrosis.
Cell death by apoptosis and necrosis in the human myocardium subjected to simulated ischaemia/reoxygenation depends on the degree of the ischaemic insult and have a different time-course with apoptosis happening early during reoxygenation and necrosis becoming more important later. Importantly, the apoptosis induced by simulated ischaemia/reoxygenation is mainly mediated by activation of caspase-3 but it does not affect necrosis.
PMCID: PMC1208913  PMID: 16111481
3.  Pharmacological and ischemic preconditioning of the human myocardium: mitoKATP channels are upstream and p38MAPK is downstream of PKC 
BMC Physiology  2002;2:10.
These studies investigate the role of mitoKATP channels, protein kinase C (PKC) and Mitogen activated protein kinase (p38MAPK) on the cardioprotection of ischemic (IP) and pharmacological preconditioning (PP) of the human myocardium and their sequence of activation.
Right atrial appendages from patients undergoing elective cardiac surgery were equilibrated for 30 min and then subjected to 90 min of simulated ischemia followed by 120 min reoxygenation. At the end of each protocol creatinine kinase leakage (CK U/g wet wt) and the reduction of MTT to formazan dye (mM/g wet wt) were measured. Similar protection was obtained with α1 agonist phenylephrine, adenosine and IP and their combination did not afford additional cardioprotection. Blockade of mitoKATP channels with 5-hydroxydecanoate, PKC with chelerythrine, or p38MAPK with SB203580 abolished the protection of IP and of PP. In additional studies, the stimulation of mitoKATP channels with diazoxide or activation of PKC with PMA or p38MAPK with anisomycin induced identical protection to that of IP and PP. The protection induced by diazoxide was abolished by blockade of PKC and by blockade of p38MAPK. Furthermore, the protection induced by PMA was abolished by SB203580 but not by 5-hydroxydecanoate, whereas the protection induced by anisomycin was unaffected by either 5-hydroxydecanoate or chelerythrine.
Opening of mitoKATP channels and activation of PKC and p38MAPK are obligatory steps in the signal transduction cascade of IP and PP of the human myocardium with PKC activation being downstream of the opening of mitoKATP channels and upstream of p38MAPK activation.
PMCID: PMC117790  PMID: 12123527

Results 1-3 (3)