Masafumi Kitakaze

Translating novel strategies for cardioprotection: the Hatter Workshop Recommendations

Ischemic heart disease (IHD) is the leading cause of death worldwide. Novel cardioprotective strategies are therefore required to improve clinical outcomes in patients with IHD. Although a large number of novel cardioprotective strategies have been discovered in the research laboratory, their translation to the clinical setting has been largely disappointing. The reason for this failure can be attributed to a number of factors including the inadequacy of the animal ischemia–reperfusion injury models used in the preclinical cardioprotection studies and the inappropriate design and execution of the clinical cardioprotection studies. This important issue was the main topic of discussion of the UCL-Hatter Cardiovascular Institute 6th International Cardioprotection Workshop, the outcome of which has been published in this article as the “Hatter Workshop Recommendations”. These have been proposed to provide guidance on the design and execution of both preclinical and clinical cardioprotection studies in order to facilitate the translation of future novel cardioprotective strategies for patient benefit.

Trials, tribulations and speculation! Report from the 7th Biennial Hatter Cardiovascular Institute Workshop

The 7th biennial Hatter Cardiovascular Institute Workshop, comprising 21 leading basic science and clinical experts, was held in South Africa in August 2012 to discuss the current cutting edge status of cardioprotection and the application of cardioprotective modalities in the clinical management of myocardial ischaemia/reperfusion injury in the context of acute coronary syndromes and cardiac surgery. The meeting, chaired by Professor Derek Yellon and Professor Lionel Opie, was run to a format of previous Hatter Cardiovascular workshops with data presented by proponents followed by discussion and debate by the faculty.

Pravastatin restored the infarct size-limiting effect of ischemic preconditioning blunted by hypercholesterolemia in the rabbit model of myocardial infarction

OBJECTIVESnWe tested to find out whether pravastatin restores the infarct size (IS)-limiting effect of ischemic preconditioning (IP) and if it has any effect on the IP-induced activation of adenosine producing enzyme ecto-5-nucleotidase which plays a key role in the IP-induced cardioprotection.nnnBACKGROUNDnThe IS-limiting effect of IP is blunted by hypercholesterolemia. Recently, HMG-CoA reductase inhibitors are shown to have direct cytoprotective effects.nnnMETHODSnRabbits were fed with a normal or cholesterol (1%) added diet with or without pravastatin (5 mg/kg/day) treatment. Infarct size was measured after 30 min occlusion and 3 h reperfusion of circumflex coronary artery with or without the IP procedure (5 min occlusion and 10 min reperfusion). Additionally, ecto-5-nucleotidase activities of ischemic and nonischemic myocardium were measured immediately after IP procedure.nnnRESULTSnThis dose of pravastatin did not normalize the increased level of serum cholesterol. The IS-limiting effect of preceding IP (IS reduced from 36.7% to 9.6%, p < 0.001) was abolished by hypercholesterolemia (from 46.1% to 31.3%, p = NS) and restored by pravastatin treatment (from 35.2% to 9.4%, p < 0.001). Pravastatin treatment did not affect IS or the effect of IP under normocholesterolemia. The activation of ecto-5-nucleotidase presented as the activity ratio of ischemic to nonischemic myocardium (3.1-fold in normocholesterolemia) was blunted by hypercholesterolemia (1.8-fold, p < 0.05) and restored by pravastatin treatment (2.9-fold).nnnCONCLUSIONSnPravastatin, at the dose serum cholesterol was not normalized, restored the IS-limiting effect of IP and IP-induced ecto-5-nucleotidase activation, which were both blunted by hypercholesterolemia. The activation of ecto-5-nucleotidase may be worth further investigation as a possible mechanism for the hypercholesterolemia-induced retardation and pravastatin-mediated restoration of the cardioprotective effect of IP.

Increased expression of P-selectin on platelets is a risk factor for silent cerebral infarction in patients with atrial fibrillation : Role of nitric oxide

BACKGROUNDnPlatelet activation and decreased levels of nitrite and nitrate (NOx), stable end products of nitric oxide (NO), are reported in patients with atrial fibrillation (AF). We examined the time-course changes in plasma NOx levels and the expression of P-selectin on platelets after the onset of AF in a canine model and determined whether these parameters could be risk factors for silent cerebral infarction in patients with AF.nnnMETHODS AND RESULTSnAF was induced by rapid atrial pacing in the canine model of AF. Plasma NOx levels were significantly decreased and the levels of P-selectin on platelets and of neutrophil/platelet conjugates were significantly increased after the onset of AF in this model. The in vitro experiments demonstrated that the inhibition of NO synthesis increased the expression of P-selectin on platelets. Plasma NOx levels (19.7+/-2.4 versus 27.5+/-2.8 micromol/L) were significantly lower in 25 patients with AF compared with age- (+/-2 years) and sex-matched control subjects. Conversely, the levels of P-selectin on platelets (7.6+/-0.8% versus 4.8+/-0.7%) and of neutrophil/platelet conjugates (14.8+/-0.9% versus 8.1+/-0.6%) were significantly higher in patients with AF. Multiple regression analysis revealed that increased P-selectin on platelets and advanced age were associated with the number of foci of silent cerebral infarction.nnnCONCLUSIONSnAn irregular heart rate that is characteristic of AF appeared to blunt NO synthesis. The increased expression of P-selectin on platelets associated with the reduced NO levels was a risk factor for silent cerebral infarction in patients with AF.

Role of Phasic Dynamism of p38 Mitogen-Activated Protein Kinase Activation in Ischemic Preconditioning of the Canine Heart

Abstract — Although ischemic stress, including ischemic preconditioning (IP), activates p38 mitogen-activated protein kinase (MAPK), the relationship between p38 MAPK activation and the underlying cellular mechanisms of cardioprotection by IP is not verified in vivo. We examined the effects of the selective p38 MAPK inhibition on the cardioprotective effect of IP in the open-chest dogs. The coronary artery was occluded 4 times for 5 minutes, separated by 5 minutes of reperfusion (IP) followed by 90 minutes of occlusion and 6 hours of reperfusion. We infused SB203580 into the coronary artery during IP and 1 hour of reperfusion, during IP alone, and during sustained ischemia in the IP group. p38 MAPK activity markedly increased during IP but did not additionally increase at the onset of ischemia and was even attenuated at 15 minutes of sustained ischemia, and heat-shock protein (HSP) 27 was phosphorylated and translocated from cytosol to myofibril or nucleus without affecting total protein level at the onset of ischemia compared with the control group. SB203580 treatment (1 &mgr;mol/L) only during IP blunted the infarct size limitation by IP (37.3±6.3% versus 7.4±2.1% in the IP group, P <0.01) and attenuated either phosphorylation or translocation of HSP27 during IP. Although the SB203580 treatment throughout the preischemic and postischemic periods had no significant effect on infarct size (33.3±9.4%) in this model, treatment with SB203580 only during ischemia partially mimicked the infarct size limitation by IP (26.8±3.5%). Thus, transient p38 MAPK activation during ischemic preconditioning mainly mediates the cardioprotection followed by HSP27 phosphorylation and translocation in vivo in the canine heart.

Nifedipine-Induced Coronary Vasodilation in Ischemic Hearts Is Attributable to Bradykinin- and NO-Dependent Mechanisms in Dogs

BACKGROUNDnDihydropyridine calcium channel blockers protect endothelial cells against ischemia and reperfusion injury, suggesting that nifedipine may increase the in vivo cardiac NO level and thus coronary blood flow (CBF) in ischemic hearts. We tested this hypothesis.nnnMETHODS AND RESULTSnIn open-chest dogs, coronary perfusion pressure (CPP) was reduced in the left anterior descending coronary artery so that CBF decreased to one third of the control level, and thereafter CPP was maintained constant (103+/-8 to 43+/-3 mm Hg, n=9). We obtained fractional shortening (FS) and lactate extraction ratio (LER) as indices of regional myocardial contraction and metabolism. Both FS (26.4+/-2.1% to 6.7+/-2.0%, n=9, P<0.001) and LER (32+/-6% to -37+/-5%, n=9, P<0.001) showed a decrease when CPP was reduced. After intracoronary infusion of nifedipine (4 microgram. kg(-1). min(-1)), CBF increased from 30+/-1 to 48+/-4 mL. 100 g(-1). min(-1) (P<0.01) without a change of CPP (n=9). Both FS (14.0+/-1.9%, n=9) and LER (-9+/-7%, n=9) also increased (P<0.01). Nifedipine increased the difference in the level of metabolites of NO (nitrate+nitrite; 9+/-3 to 25+/-5 nmol/mL, n=9, P<0.01) and bradykinin (22+/-5 to 58+/-4 pmol/mL, n=9, P<0.01) between coronary venous and arterial blood. L-NAME (an NO synthase inhibitor) or HOE-140 (a bradykinin receptor antagonist) attenuated (P<0.05) the increase in CBF (29+/-3 and 35+/-2 mL. 100 g(-1). min(-1), n=5 each), FS (4.8+/-0.6% and 6.9+/-1.7%, n=5 each), LER (-47+/-8% and -35+/-9%, n=5 each), and nitrate+nitrite (3+/-2 and 8+/-4 nmol/mL, n=5 each) due to nifedipine infusion.nnnCONCLUSIONSnThese results indicate that the calcium channel blocker nifedipine mediates coronary vasodilation and improves myocardial ischemia through both bradykinin/NO-dependent and -independent mechanisms.

NITRIC OXIDE MEDIATES PROTEIN KINASE C ISOFORM TRANSLOCATION IN RAT HEART DURING POSTISCHEMIC REPERFUSION

It is controversial whether nitric oxide (NO) is protective or deleterious against ischemia-reperfusion injury. We examined the effect of NO on PKC isoform translocation and protection against ischemia-reperfusion injury in perfused heart. An NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester, 3.0 microM), administered only during reperfusion but not during ischemia, inhibited the translocation of PKC-alpha, -delta and -epsilon isoforms to the nucleus-myofibril fraction and the translocation of PKC-alpha to the membrane fraction after ischemia (20 min) and reperfusion (10 min) in the perfused rat heart. NO donors, 3-morpholinosydnonimine (SIN-1) or S-nitroso-N-acetylpenicillamine (SNAP) activated purified PKC in vitro. SIN-1 also induced PKC isoform translocation in perfused heart. On the other hand, PKC selective inhibitor, calphostin C (0.2 microM) or chelerythrine (1.0 microM), aggravated the contractile dysfunction of ischemic heart during reperfusion, when they were perfused during reperfusion. These data suggest that NO generated during reperfusion following ischemia activates PKC isoforms and may protect the heart against contractile dysfunction in the perfused rat heart.

Amelioration of ischemia- and reperfusion-induced myocardial injury by the selective estrogen receptor modulator, raloxifene, in the canine heart.

OBJECTIVESnWe sought to investigate whether raloxifene reduces ischemia-reperfusion injury and what mechanisms are involved in the cardioprotective effects.nnnBACKGROUNDnEstradiol-17-beta reduces myocardial infarct size in ischemia-reperfusion injury. Raloxifene, a selective estrogen receptor modulator, demonstrates immediate coronary artery vasorelaxing effects.nnnMETHODSnThe myocardial ischemia-reperfusion model included anesthetized open-chest dogs after 90-min occlusion of the left anterior descending coronary artery (LAD) and subsequent 6-h reperfusion. Raloxifene and/or other drugs were infused into the LAD from 10 min before coronary occlusion to 1 h after reperfusion without an occlusion period.nnnRESULTSnInfarct size was reduced in the raloxifene (5 microg/kg per min) group compared with the control group (7.2 +/- 2.5% vs. 40.9 +/- 3.9% of the area at risk, p < 0.01). Either N(G)-nitro-L-arginine methyl ester (L-NAME), the inhibitor of nitric oxide (NO) synthase, or charybdotoxin, the blocker of Ca(2+)-activated K+ (K(Ca)) channels, partially attenuated the infarct size-limiting effect, and both of them completely abolished the effect. The incidence of ventricular fibrillation was also less in the raloxifene group than in the control group (11% vs. 44%, p < 0.05). Activity of p38 mitogen-activated protein (MAP) kinase increased with 15-min ischemia, and raloxifene pretreatment inhibited the activity. Myeloperoxidase activity of the 6-h reperfused myocardium was also attenuated by raloxifene.nnnCONCLUSIONSnThese data demonstrate that raloxifene reduces myocardial ischemia-reperfusion injury by mechanisms dependent on NO and the opening of K(Ca) channels in canine hearts. Deactivation of p38 MAP kinase and myeloperoxidase by raloxifene may be involved in the cellular mechanisms of cardioprotection.

Adenosine therapy: a new approach to chronic heart failure

Both the prevention and attenuation of chronic heart failure (CHF) are important issues for cardiologists. There are three different strategies to prevent patients from deleterious sequels. The first strategy is to remove the causes of CHF if possible; the second is to attenuate the events that may lead to CHF, such as myocardial ischaemia and reperfusion injury, cardiomyopathy and myocarditis, cardiac hypertrophy and ventricular remodelling; the third is to prevent or attentuate the progression of CHF. Adenosine has a number of actions which merit it as a possible cardioprotective and therapeutic agent for CHF. Firstly, adenosine induces collateral circulation via inducing growth factors and triggering ischaemic preconditioning, both of which induce ischaemic tolerance in advance. Adenosine is also known to reduce the release of noradrenaline, production of endothelin and attenuate the activation of renin-angiotensin system all of which are believed to cause cardiac hypertrophy and remodelling. Secondly, exogenous adenosine is known to reduce the severity of ischaemia and reperfusion injury. Thirdly, adenosine is reported to counteract neurohumoral factors, i.e., cytokine systems, known to be related to the pathophysiology of CHF. Recently, we revealed that adenosine metabolism is changed in patients with CHF and increases in adenosine levels may aid to reduce the severity of CHF. Thus, there are many potential mechanisms for cardioprotection attributable to adenosine and we postulate the use of adenosine therapy will be beneficial in patients with CHF.

Plasma adenosine levels and platelet activation in patients with atrial fibrillation.

Platelet activation is observed in patients with atrial fibrillation (AF). P-selectin, which is expressed on platelet activation, plays an important role in the formation of thromboemobli. Because adenosine is known to attenuate platelet activation, we evaluated adenosine levels and 2 indicators of platelet activation, i.e., expression of P-selectin on platelets and plasma levels of beta-thromboglobulin, in 28 patients with AF (20 men and 8 women, age range 64+/-2 years) with sex- and age-matched (+/-2 years) subjects with sinus rhythm. The incidence of risk factors for stroke except for coronary heart disease and in echocardiographic parameters did not differ between the 2 groups. Plasma adenosine levels were lower (p <0.05) in patients with AF than in controls (mean [interquartile range] 13.4 [19.3-9.3] vs 19.1 [30.8-11.9] nmol/L). The expression of P-selectin on platelets (6.8% [13.6-3.4] vs 4.0% [8.8-1.8]) and plasma levels of beta-thromboglobulin were higher (p <0.05) in patients with AF. Flow cytometric analysis revealed that an antagonist of adenosine receptors, 8-sulfophenyltheophylline, increased the expression of P-selectin on platelets in a dose-dependent manner in the in vitro study. These results suggest that decreased plasma levels of adenosine were associated with platelet activation in patients with AF. Substitution of adenosine may provide a strategy for preventing platelet activation in these patients.