Ben C.G. Gho

Rapid Ventricular Pacing Produces Myocardial Protection by Nonischemic Activation of KATP+ Channels

BACKGROUND Rapid ventricular pacing reduces the incidence of ventricular arrhythmias during a subsequent sustained period of ischemia and reperfusion. We investigated whether rapid ventricular pacing also limits myocardial infarction and determined the role of KATP+ channels in the protection afforded by ventricular pacing. METHODS AND RESULTS Myocardial infarction was produced by a 60-minute coronary artery occlusion in open chest pigs. Infarct size of pigs subjected to 10 minutes of ventricular pacing at 200 beats per minute followed by 15 minutes of normal sinus rhythm before the occlusion (79 +/- 3% of the area at risk, mean +/- SEM) was not different from control infarct size (84 +/- 2%). Thirty-minute pacing followed by 15-minute sinus rhythm resulted in modest reductions in infarct size (71 +/- 2%, P<.05 versus control). Thirty minutes of pacing immediately preceding the occlusion without intervening sinus rhythm resulted in considerable limitation of infarct size (63 +/- 4%, P<.05), which was abolished by pretreatment with the KATP+ channel blocker glibenclamide (78 +/- 4%, P=NS). KATP+ channel activation did not appear to involve ischemia: (1) myocardial endocardial/epicardial blood flow ratio was 1.07 +/- 0.08, (2) phosphocreatine and ATP levels and arterial-coronary venous differences in pH and PCO2 were unchanged, (3) end-systolic segment length did not increase and postsystolic shortening was not observed during pacing, and (4) systolic shortening recovered immediately to baseline levels and coronary reactive hyperemia was absent after cessation of pacing. Administration of glibenclamide after 30 minutes of pacing at the onset of 15 minutes of normal sinus rhythm did not attenuate the protection (73 +/- 3%, P<.05 versus control), suggesting the KATP+ channels did not contribute to the moderate degree of protection that was still present 15 minutes after cessation of pacing. CONCLUSIONS Rapid ventricular pacing protects the myocardium against infarction via nonischemic KATP+ channel activation. Continued activation of KATP+ channels does not appear mandatory for the protection that is still present 15 minutes after cessation of pacing.

Hypothermia extends the cardioprotection by ischaemic preconditioning to coronary artery occlusions of longer duration

OBJECTIVE To test the hypothesis that mild hypothermia potentiates the cardioprotection afforded by ischaemic preconditioning so that infarct size limitation can be obtained after coronary artery occlusion (CAO) durations which exceed the cardioprotective range (> 90 min) of either hypothermia or ischaemic preconditioning alone. METHODS Four groups of anaesthetized rats were subjected to different durations of CAO: (i) normothermia (N, 36.5-37.5 degrees C, n = 29), (ii) normothermia + ischaemic preconditioning (N + IP, 15 min CAO followed by 10 min of reperfusion, n = 35), (iii) hypothermia (H, 30-31 degrees C, n = 31) and (iv) hypothermia + ischaemic preconditioning (H + IP, n = 24). Infarct size (IA/AR) was determined after 3 hours of reperfusion using trypan blue to delineate the area at risk (AR) from non-risk region and nitroblue tetrazolium to delineate infarcted area (IA) from viable myocardium. RESULTS In N the CAO duration versus infarct size relation had a sigmoid shape with virtually no infarction occurring at 15 min CAO and 56 +/- 5% of the area at risk being infarcted at 30 min CAO reaching a plateau of 71 +/- 2% at 60 min CAO. Hypothermia produced a rightward shift of the relation resulting in an approximately 15 min delay in onset of infarction. Ischaemic preconditioning produced a similar reduction in infarct size (23 +/- 4%) at 30 min CAO compared to hypothermia (13 +/- 3%) but also limited infarct size at 45 min to 36 +/- 3% and at 60 min CAO to 50 +/- 3% suggesting a slowing of infarct progression. Neither intervention limited IA/AR produced by 120 min CAO. In H + IP, combined hypothermia and ischaemic preconditioning resulted in synergistic infarct size reduction so that at 45 min and 60 min CAO IA/AR was reduced to 17 +/- 3% and 23 +/- 3%, respectively, and even at 120 min CAO to 58 +/- 5%, which was significantly smaller than during normothermic control conditions (p < 0.05 vs. N). CONCLUSION Mild hypothermia limited IA/AR modestly but markedly enhanced the cardioprotection afforded by ischaemic preconditioning in the in situ rat heart so that irreversible damage produced by even prolonged coronary artery occlusions was limited.

Cardioprotection by ischemic and nonischemic myocardial stress and ischemia in remote organs. Implications for the concept of ischemic preconditioning.

Ischemic preconditioning studies employ one or more brief total coronary artery occlusions separated by complete reperfusion to limit infarct size during a subsequent prolonged coronary artery occlusion. We now present evidence that in anesthetized pigs a partial coronary artery occlusion without intervening reperfusion between the partial and prolonged total occlusions can also precondition the myocardium provided that the reduction in coronary blood flow is sufficiently severe. Thus infarct size was reduced after a 60 min total coronary artery occlusion when the total occlusion was preceded by a partial coronary occlusion that reduced coronary blood flow by 70% but not when the flow reduction was only 30%. In this two-stage coronary occlusion model the degree of protection appears greater in the epicardial than in the endocardial half. In view of evidence that brief occlusions of a coronary artery also protect myocardium outside its perfusion territory, we subsequently investigated whether ischemia in remote organs can protect myocardium. Because of reports that development of infarct size may be temperature dependent, we also investigated whether the cardioprotection by remote organ ischemia was temperature dependent. In anesthetized rats a 15 min coronary artery occlusion was more effective in reducing infarct size produced by a subsequent 60 min total coronary artery occlusion when the experiments were performed at a body core temperature of 30-31 degrees C than at 36-37 degrees C, while infarct size of animals which were subjected to only the 60 min total coronary artery occlusion was the same for the two body core temperatures. In rats with a body core temperature of 36-37 degrees C a 15 min mesenteric artery occlusion, but not a 15 min renal artery occlusion, reduced infarct size produced by a subsequent 60 min coronary artery occlusion. When the experiments were performed at 30-31 degrees C both the mesenteric and renal artery occlusions were protective. These observations indicate the local myocardial ischemia is not required to protect the myocardium during a prolonged coronary occlusion. We further investigated whether myocardium could also be protected by a cardiac stimulus which does not produce ischemia at all. For this purpose we electrically paced the left ventricle of anesthetized pigs to produce heart rates of 200 bpm (which did not lead to ischemia as assessed by a number of functional and biochemical variables) and found that 30 min of ventricular pacing reduced myocardial infarct size produced by a subsequent 60 min coronary artery occlusion. The protection by ventricular pacing involved activation of K+ATP channels as pretreatment with glibenclamide abolished the protection by ventricular pacing. We conclude that a number of distinctly different stimuli can protect the myocardium suggesting that ischemic myocardial preconditioning could be just one feature of a more general protection phenomenon.

Nucleotide sequence and expression of the porcine vascular endothelial growth factor

We cloned and sequenced two cDNAs encoding the angiogenic, vascular endothelial growth factor (VEGF) from the porcine heart. Deduced amino acid sequence of the clone pPVE-18 and pPVE-5 predicted 164 (VEGF164), and 120 (VEGF120) residues of VEGF, respectively, with a putative N-terminal signal sequence of 26 amino acids. The porcine VEGF is shorter by one amino acid as compared to human VEGF, but a potential glycosylation site is present at Asn-74. PCR detection, and verification of the identity of the PCR products by Southern hybridization, confirmed wide expression of VEGF in different porcine tissues. Northern blot analysis with a radiolabeled porcine specific VEGF probe, showed one major (3.9 kb) and one minor (1.7 kb) mRNA species expressed in all four chambers of the heart.

Endocardial and epicardial infarct size after preconditioning by a partial coronary artery occlusion without intervening reperfusion. Importance of the degree and duration of flow reduction

OBJECTIVE Recently, we reported that a partial coronary artery occlusion immediately preceding a sustained coronary artery occlusion limited infarct size. We now investigated whether the protection by partial coronary artery occlusions (i) depends on the severity and(or) duration of the flow reduction and (ii) varies in the different myocardial layers. METHODS In 71 open-chest pigs (eight groups) left ventricular area at risk (AR) and infarct area (IA) were determined for the endocardial (IAendo and ARendo) and epicardial halves (IAepi and ARepi). RESULTS In control animals (60 min total coronary artery occlusion (TCO) followed by 120 min reperfusion (Rep)) there were highly linear relations between IA and AR in the endocardium (r = 0.98, P < 0.01) and epicardium (r = 0.97, P < 0.01), which could be described by IAendo = 1.01 ARendo - 4.5 and by IAepi = 0.88ARepi - 3.6, respectively. In animals that underwent a 10 min TCO + 15 min Rep prior to the 60 min TCO + 120 min Rep, IA in both myocardial layers were again highly linearly related with AR, with less steep slopes for both the endocardium (0.63) and epicardium (0.57) (both P < 0.01). Two groups of pigs were subjected to either a 30 or 90 min 70% reduction in coronary blood flow (FR) immediately preceding the 60 min TCO + 120 min Rep, without intervening reperfusion. A 30 min 70% FR decreased IA to the same degree in the endo- and epicardial half. A 90 min 70% FR resulted in protection in the epicardium (P < 0.01) but not in the endocardium, most likely because 90 min 70% FR without 60 min TCO already caused infarction which was more severe in the endo- than in the epicardium (P < 0.01). Endocardial and epicardial IA after either a 30 or 90 min 30% FR prior to the 60 min TCO was not different from that in the control group, indicating that this mild flow reduction failed to limit irreversible damage. CONCLUSIONS Thirty or ninety min of severe (70%) but not mild (30%) coronary flow reductions protected against myocardial infarction. The protection by a 70% FR was influenced by the duration of FR as a 30 min 70% FR similarly decreased IA in the endocardial and epicardial halves, while 90 min 70% FR preferentially limited IA in the epicardial half. These findings suggest that perfusion abnormalities immediately preceding an infarction could be an important source of infarct size variability in patients.

Haemodynamic profile of the potassium channel activator EMD 52692 in anaesthetized pigs.

1 The systemic and regional haemodynamic effects of the potassium channel activator EMD 52692 or its solvent were investigated after intravenous and after intracoronary administration in anaesthetized pigs. 2 Consecutive intravenous 10 min infusions of EMD 52692 (0.15, 0.30, 0.60, 1.20 μg kg−1 min−1; n = 7) dose‐dependently decreased mean arterial blood pressure by up to 50%. This was entirely due to peripheral vasodilatation, since cardiac output did not change. Heart rate increased by up to 50%, while left ventricular end diastolic pressure decreased dose‐dependently from 6 ± 1 mmHg to 3 ± 1 mmHg (P > 0.05), and stroke volume decreased from 30 ± 2 ml to 21 ± 2 ml (P > 0.05). Left ventricular dP/dtmax was not affected. 3 Although cardiac output did not change, EMD 52692 caused a redistribution of blood flow from the arteriovenous anastomoses to the capillary channels. Blood flow to the adrenals, small intestine, stomach, bladder, spleen and brain increased, while renal blood flow decreased and blood flow to several muscle groups and skin were not altered. Vascular conductance was increased dose‐dependently in all organs, except for the kidneys, where after the initial increase, vascular conductance returned to baseline with the highest dose. Particularly striking were the effects on the vasculature of the brain. With the highest dose of EMD 52692 blood flow more than doubled, while vascular conductance increased four fold. 4 Transmural myocardial blood flow increased slightly, which was entirely due to an increase in subepicardial blood flow. Myocardial O2‐consumption and segment length shortening were not significantly affected. 5 After consecutive 10 min intracoronary infusions (0.0095, 0.019, 0.0375 and 0.075 μg kg−1 min−1; n = 7) into the left anterior descending coronary artery (LADCA), mean arterial blood pressure was maintained with the lowest two doses, but decreased by up to 15% with the higher doses, whereas heart rate increased by up to 24%. Blood flow to the LADCA‐perfused myocardium doubled with the highest dose, the subepicardium benefitting the most. Coronary venous O2‐saturation increased dose‐dependently from 23 ± 2% to 60 ± 4%, while myocardial O2‐consumption of the LADCA‐perfused myocardium was not affected by the drug. 6 It is concluded that EMD 52692 is a potent vasodilator, with particularly pronounced effects on vasculature of the brain. Its selectivity for vascular smooth muscle cells exceeds that for the myocytes, since with doses that are much higher than those of potential clinical interest no negative inotropic effects were observed. The compound primarily dilates arteries but some venodilatation may also occur.

Effect of antiischemic therapy on coronary flow reserve and the pressure-maximal coronary flow relationship in anesthetized swine.

The effect Of nifedipine (0.5, 1.0, and 2.0 μg/kg/min); metoprolol (0.1, 0.5, and 1.0 mg/kg), the (β1-selective adrenoceptor partial agonist epanolol (10, 50, and 200 μ.g/kg), or equivalent volumes of isotonic saline (n = 6. in each group), on coronary blood flow capacity were studied in anesthetized swine.Intracoronary bolus injections of adenosine (20 μg/kg/10.2 ml) were administered without and during three levels of coronary stenosis, prior to and following each dose of drug, to obtain maximal coronary blood flows at different perfusion pressures in the autoregulatoryrange. Coronary perfusion pressures were varied by partial inflation of a balloon around the left anterior descending coronary artery. Special care was taken that the stenoses not lead to myocardial ischemia. Three indices of coronary blood flow capacity were used: absolute coronary flow reserve (ACFR, the ratio of maximal to resting coronary blood flow). the slope and the extrapolated pressure at zero flow (P2f) of the pressure-maximal coronary flow (PMCF) relationship, and relative coronary flow reserve (RCFR, the ratio of maximal coronary blood flow with a stenosis to maximal coronary blood flow without a stenosis) at two of the three levels of stenosis. Nifedipine decreased ACFR from 4.5 + 1.9 to 1.9 + 0.3 (mean ± SD: p < 0.05), reflecting in part the increase in resting coronary blood flow. The nifedipine-induced changes in maximal coronary blood flow were not only due to a drop in perfusion pressure, as the slope of the PMCF relationship decreased from 2.27 + (49 m1/(min.mm Hg) to 1.54 + 0.51 mll(min.mm Hg) (p < 0.05), and P2f decreased from 30 ± 4 mm Hg to 20 ± 7 mm Hg (p < 0.05). Consequently, calculated maximal coronary blood flow was attenuated from 114 31 ml/min to 93 ± 37 ml/min at 80 mm Hg; but was enhanced from 23 ± 13 to 37 + 24 ml/min at 40 mm Hg coronary perfusion pressure. In concert with the change in the PMCF relationship. RCFR at equivalent severe stenosis increased from 0.33 ± 0.06 to 0.47 ± 0.10 (p < 0.05). No changes were observed with metoprolol, epanolol, or saline. The effect of nifedipine on the PMCF relationship not only provides a mechanism for the drugs anti ischemic action, but should also he considered in the interpretation of coronary flow reserve measurements in patients on nifedipine treatment.

CARDIOPROTECTION BY ORGANS IN STRESS OR DISTRESS

ConclusionSeveral studies now suggest that the myocardium can be protected by forms of stress other than myocardial ischemia. These include stimuli which stress the heart but do not lead to ischemia (stretch ventricular pacing) and stimuli which cause distress (ischemia) in organs other than the heart. Since ischemia in remote organs such as kidney (14), brains (15), skeletal muscle (16) and liver (17) can also precondition these organs, it is clear that several forms of stress or distress are capable of triggering processes which protect the organ subjected to the stimulus as well as other organs. It would thus appear that ischemic myocardial preconditioning may be merely one aspect of a general phenomenon that results in organ protection. Future investigations should therefore aim to unravel the mechanisms leading to organ protection which may then be pharmacologically exploited. It is also important to keep in mind, however, that not all forms of (di)stress (myocardial hibernation (4), smoking (18) result in myocardial protection. Finally, these newly recognized protective stimuli may, in addition to problems with infarct size determination in patients as outlined elsewhere (19, 20), be confounding factors in finding definitive proof for the occurrence of ischemic myocardial preconditioning in man.

Novel Approaches to Myocardial Preconditioning in Pigs

The ability of myocardium to adapt to ischemic stress has already been documented in both experimental and clinical studies employing intermittent ischemia some 15 years ago. For instance, it was shown that changes in metabolic markers of myocardial ischemia were much less during the second of two consecutive, but identical coronary blood flow reductions in pigs.1 Similarly, the second of two consecutive but identical atrial pacing stress tests in patients undergoing a cardiac catheterization for suspected coronary artery disease showed less severe signs of ischemia.2 At that time, no attempt was made to elucidate the mechanism of the altered response during the second episode of ischemia as the purpose of these studies was to develop models of repeated reversible ischemia with reproducible changes in the various markers of ischemia. In such models it would then be possible to evaluate the effects of pharmacological interventions with the animal or patient serving as its own control. Several subsequent studies on the metabolic and functional consequences of multiple brief periods of ischemia also revealed the absence of cumulative losses in tissue levels of high energy phosphates and in regional contractile function, but these have not always been consistent findings.3

Myocardial Protection by Brief Ischemic and Nonischemic Stress

Publisher Summary This chapter presents an overview of myocardial protection by brief ischemic and nonischemic stress. The chapter reviews the evidence for protection by ischemic preconditioning in other organs than the heart. Ischemic preconditioning has also been shown to occur in the lung of spontaneously breathing cats. In these animals, lung pathology was assessed after two hours of reperfusion following two hours of ischemia. Ischemic preconditioning with 10 minutes of ischemia and 10 minutes of reperfusion limited edematous alveoli to seven percent as compared to 22% in the control group. The data of the preconditioned lungs were similar to those observed after two hours of ischemia and one hour of reperfusion, suggesting that preconditioning limited reperfusion injury rather than ischemic injury, and that a longer duration of reperfusion may be necessary to fully assess damage. Evidence is also accumulating that transient ischemia induces tolerance and protects the brain from subsequent ischemia. Ischemic preconditioning by partial coronary artery occlusion without intervening reperfusion is also described in the chapter.