Sandra de Zeeuw

Time Course and Mechanism of Myocardial Catecholamine Release During Transient Ischemia In Vivo

BACKGROUND Elevated concentrations of norepinephrine (NE) have been observed in ischemic myocardium. We investigated the magnitude and mechanism of catecholamine release in the myocardial interstitial fluid (MIF) during ischemia and reperfusion in vivo through the use of microdialysis. METHODS AND RESULTS In 9 anesthetized pigs, interstitial catecholamine concentrations were measured in the perfusion areas of the left anterior descending coronary artery (LAD) and the left circumflex coronary artery. After stabilization, the LAD was occluded for 60 minutes and reperfused for 150 minutes. During the final 30 minutes, tyramine (154 nmol. kg(-1). min(-1)) was infused into the LAD. During LAD occlusion, MIF NE concentrations in the ischemic region increased progressively from 1. 0+/-0.1 to 524+/-125 nmol/L. MIF concentrations of dopamine and epinephrine rose from 0.4+/-0.1 to 43.9+/-9.5 nmol/L and from <0.2 (detection limit) to 4.7+/-0.7 nmol/L, respectively. Local uptake-1 blockade attenuated release of all 3 catecholamines by >50%. During reperfusion, MIF catecholamine concentrations returned to baseline within 120 minutes. At that time, the tyramine-induced NE release was similar to that seen in nonischemic control animals despite massive infarction. Arterial and MIF catecholamine concentrations in the left circumflex coronary artery region remained unchanged. CONCLUSIONS Myocardial ischemia is associated with a pronounced increase of MIF catecholamines, which is at least in part mediated by a reversed neuronal reuptake mechanism. The increase of MIF epinephrine implies a (probably neuronal) cardiac source, whereas the preserved catecholamine response to tyramine in postischemic necrotic myocardium indicates functional integrity of sympathetic nerve terminals.

Cardiac interstitial fluid levels of angiotensin I and II in the pig.

OBJECTIVE To study whether cardiac interstitial fluid levels of angiotensin I and II (Ang I and II) can be monitored in vivo, using the microdialysis technique, and to assess the contribution of plasma-derived angiotensins to the interstitial fluid levels of these peptides. DESIGN AND METHODS Microdialysis probes were placed in the left ventricular (LV) myocardium of eight anaesthetized pigs, three of which were untreated and five treated with the angiotensin II type 1 (AT1) receptor antagonist L-158,809 (10 mg intracoronary). All pigs were given a 1 h intracoronary infusion of 125I-Ang II. Aortic and coronary venous blood samples were taken under steady-state conditions, and interstitial dialysate was collected during the entire infusion period. Immediately after stopping the infusion, LV tissue pieces were obtained at various time points. RESULTS L-158,809 did not affect the levels of endogenous Ang I and II or the levels of plasma 125I-Ang II. Aortic Ang I and II levels (22 and 16 fmol/ml; geometric mean of eight pigs) were comparable to coronary venous Ang I and II levels, whereas the coronary venous 125I-Ang II levels (6650 c.p.m./ml) were approximately 30 times higher than those in the aorta. Tissue Ang I and II levels were 5 and 17 fmol/g, respectively. In untreated animals, the 125I-Ang II levels per g LV tissue were similar to the levels per ml coronary venous plasma, and the ex vivo half-life of tissue 1251-Ang II was > 30 min. In treated animals, tissue 125I-Ang II was < 5% of coronary venous 125I-Ang II and became undetectable within 15 min. 125I-Ang II, Ang I and Ang II levels in the interstitial fluid were close to or below the detection limit (200 c.p.m., 60 fmol and 20 fmol per ml, respectively) in all animals. CONCLUSIONS Plasma and myocardial interstitial fluid angiotensin levels are of the same order of magnitude. Plasma Ang II does not contribute to the interstitial fluid level of Ang II, most likely because of its rapid metabolism in the vascular wall. Binding to AT1 receptors protects Ang II against metabolism.

Role of adenosine in ischemic preconditioning in rats depends critically on the duration of the stimulus and involves both A1 and A3 receptors

OBJECTIVES There is currently general agreement that adenosine is not involved in ischemic preconditioning (IP) in rat hearts. We hypothesized that the failure to show a role for adenosine is due to the use of brief preconditioning stimuli, and therefore investigated whether adenosine is involved when longer stimuli are employed and which receptor subtypes are involved. METHODS AND RESULTS Infarct size (IS) was determined in anesthetized rats after 180 min of reperfusion (REP) following a 60-min coronary artery occlusion (CAO). IS was 69+/-2% (n=15) of the risk area in control rats and 45+/-2% (n=19; P<0.05) following IP by a single 15-min CAO. The non-selective adenosine receptor antagonist SPT, which itself had no effect on IS (74+/-1%), blunted the protection by IP (IS=57+/-2%, P<0.05) in a dose of 2 x 5 mg/kg i.v., and abolished the protection (IS=70+/-1%) at 2 x 25 mg/kg i.v. Following IP by three cycles of 3-min CAO and 3-min REP, IS was 24+/-6% (P<0.05), which was not affected by SPT in doses of 2 x 10 and 2 x 25 mg/kg i.v. The A(3) antagonist MRS-1191 (3.3 mg/kg, i.p.), which itself did not affect IS (70+/-2%), blunted the protection by IP with a 15-min CAO (IS=54+/-2%, P<0.05). When 2 x 5 mg/kg SPT (a dose selective for A(1)-receptors, as it did not affect the protection by the A(3) selective agonist IB-MECA, 51+/-3%) and MRS 1191 were combined the protection by IP was abolished (IS=67+/-2%). CONCLUSIONS Involvement of adenosine in IP in rats depends critically on the duration of the stimulus. Thus, whereas adenosine was not involved when stimuli of 3-min duration were employed, activation of both A(1) and A(3) receptors contributed when a stimulus of 15 min was used.

Cardioprotection in Pigs by Exogenous Norepinephrine but not by Cerebral Ischemia–Induced Release of Endogenous Norepinephrine

Background and Purpose— Endogenous norepinephrine release induced by cerebral ischemia may lead to small areas of necrosis in normal hearts. Conversely, norepinephrine may be one of the mediators that limit myocardial infarct size by ischemic preconditioning. Because brief ischemia in kidneys or skeletal muscle limits infarct size produced by coronary artery occlusion, we investigated whether cardiac norepinephrine release during transient cerebral ischemia also elicits remote myocardial preconditioning. Methods— Forty-one crossbred pigs of either sex were assigned to 1 of 7 experimental groups, of which in 6 groups myocardial infarct size was determined after a 60-minute coronary occlusion and 120 minutes of reperfusion. One group served as control (no pretreatment), while the other groups were pretreated with either cerebral ischemia or an intracoronary infusion of norepinephrine. Results— In 10 anesthetized control pigs, infarct size was 84±3% (mean±SEM) of the area at risk after a 60-minute coronary occlusion and 120 minutes of reperfusion. Intracoronary infusion of 0.03 nmol/kg · min−1 norepinephrine for 10 minutes before coronary occlusion did not affect infarct size (80±3%; n=6), whereas infusion of 0.12 nmol/kg · min−1 limited infarct size (65±2%; n=7;P <0.05). Neither 10-minute (n=5) nor 30-minute (n=6) cerebral ischemia produced by elevation of intracranial pressure before coronary occlusion affected infarct size (83±4% and 82±3%, respectively). Myocardial interstitial norepinephrine levels tripled during cerebral ischemia and during low-dose norepinephrine but increased 10-fold during high-dose norepinephrine. Norepinephrine levels increased progressively up to 500-fold in the area at risk during the 60-minute coronary occlusion, independent of the pretreatment, while norepinephrine levels remained unchanged in adjacent nonischemic myocardium and arterial plasma. Conclusions— Cerebral ischemia preceding a coronary occlusion did not modify infarct size, which is likely related to the modest increase in myocardial norepinephrine levels during cerebral ischemia. The infarct size limitation by high-dose exogenous norepinephrine is not associated with blunting of the ischemia-induced increase in myocardial interstitial norepinephrine levels.

New Insights into Cardioprotection by Ischemic Preconditioning and Other Forms of Stressa

Abstract: Ischemic preconditioning has not only received wide attention in heart research, but has also been a topic of extensive studies involving other organs. In several of these studies, it has been shown that in spite of differences in the endpoints used to assess protection, the same mediators as in myocardial ischemic preconditioning may be involved. However, several of the putative mediators do not require ischemia to become activated. This has guided us and others to investigate whether the myocardium can also be protected by brief ischemia in other organs and whether other non‐pharmacological forms of stress, which do not produce ischemia but are capable of activating these potential mediators, are also cardioprotective.

Epinephrine in the Heart Uptake and Release, but No Facilitation of Norepinephrine Release

Background—Several studies have suggested that epinephrine augments the release of norepinephrine from sympathetic nerve terminals through stimulation of presynaptic receptors, but evidence pertaining to this mechanism in the heart is scarce and conflicting. Using the microdialysis technique in the porcine heart, we investigated whether epinephrine, taken up by and released from cardiac sympathetic nerves, can increase norepinephrine concentrations in myocardial interstitial fluid (NEMIF) under basal conditions and during sympathetic activation. Methods and Results—During intracoronary epinephrine infusion of 10, 50, and 100 ng/kg per minute under basal conditions, large increments in interstitial (from 0.31±0.05 up to 140±30 nmol/L) and coronary venous (from 0.16±0.08 up to 228±39 nmol/L) epinephrine concentrations were found, but NEMIF did not change. Left stellate ganglion stimulation increased NEMIF from 3.4±0.5 to 8.2±1.5 nmol/L, but again, this increase was not enhanced by concomitant intracoronary epinephrine infusion. Intracoronary infusion of tyramine resulted in a negligible increase in epinephrine concentration in myocardial interstitial fluid (EPIMIF), whereas 30 minutes after infusion of epinephrine an increase of 9.5 nmol/L in EPIMIF was observed, indicating that epinephrine is taken up by and released from cardiac sympathetic neurons. Although 68% to 78% of infused epinephrine was extracted over the heart, the ratio of interstitial to arterial epinephrine concentrations was only ≈20%, increasing to 29% with neuronal reuptake inhibition. Conclusions—Our findings demonstrate epinephrine release from cardiac sympathetic neurons, but they do not provide evidence that epinephrine augments cardiac sympathoneural norepinephrine release under basal conditions or during sympathetic activation.

Cardiovascular profile of the calcium sensitizer EMD 57033 in open-chest anaesthetized pigs with regionally stunned myocardium

Ca2+ sensitizers enhance systolic function, but may impair relaxation in vitro; these effects may differ in stunned and normal myocardium. We therefore studied the effect of EMD 57033 on systolic and diastolic function of normal and stunned porcine myocardium in vivo. Myocardial stunning by 15 min coronary occlusion and 30 min reperfusion abolished systolic shortening (SS) (baseline 13±1%) and decreased end‐systolic elastance (Ees) from 67±7 to 47±5 mmHg mm−1 (both P<0.05). Maximum rate of fall of myocardial elastance (dE/dtmin) decreased from −850±100 to −320±30 mmHg mm−1 s−1, while the time constant τe of the decay of elastance increased from 58±3 to 68±6 ms (both P<0.05). End‐diastolic elastance (Eed) was unchanged although the zero pressure intercept (L0,ed) had increased. In the stunned region, EMD 57033 (0.2 mg kg−1 min−1 for 60 min, i.v., n=7) increased SS to 19±2%, Ees to 287±40 mmHg mm−1, dE/dtmin to −3630±640 mmHg mm−1 s−1 and decreased τe to 50±3 ms, while Eed remained unchanged. In the normal region, EMD 57033 increased SS from 14±2 to 18±3%, Ees from 59±4 to 263±23 mmHg mm−1, dE/dtmin from −480±70 to −2280±700 mmHg mm−1 s−1 and decreased τe from 91±12 to 61±3 ms (all P<0.05), while Eed remained unchanged. These responses were minimally affected by adrenoceptor blockade (n=7). Vehicle (n=7) had no effect on either region. EMD 57033 increased cardiac output (up to 27±8%) and LVdP/dtmax (86±19%). Mean aortic pressure decreased (19±7%) due to systemic vasodilation that was not amenable to blockade of adrenoceptors or NO synthesis. In conclusion, EMD 57033 restored systolic and diastolic function of stunned myocardium, and produced similar improvements in systolic and diastolic function in normal myocardium.

Does protein kinase C play a pivotal role in the mechanisms of ischemic preconditioning

SummaryThis communication reviews the evidence for the pivotal role of protein kinase C in ischemic myocardial preconditioning. It is believed that several intracellular signalling pathways via receptor-coupled phospholipase C and its ‘cross-talk” with phospholipase D converge to activation of protein kinase C isotypes which is followed by phosphorylation of until now (a number of) unknown target proteins which produce the protective state of ischemic preconditioning.After briefly introducing the general biochemical properties of protein kinase C, its isotypes and the limitations of the methodology used to investigate the role of protein kinase C, studies are discussed in which pharmacological inhibition and activation and (immunore)activity and/or isotypes measurements of protein kinase C isotypes were applied to assess the role of activation of protein kinase C in ischemic myocardial preconditioning.It is concluded that definitive proof for the involvement of protein kinase C in preconditioning requires future studies which must focus on the isotype(s) of protein kinase C that are activated, the duration of action, cellular translocation sites and the identity and stability (of covalently bound phosphate) of phosphorylated substrate proteins.

Exogenous angiotensin II does not facilitate norepinephrine release in the heart

Abstract—Studies on the effect of angiotensin II on norepinephrine release from sympathetic nerve terminals through stimulation of presynaptic angiotensin II type 1 receptors are equivocal. Furthermore, evidence that angiotensin II activates the cardiac sympathetic nervous system in vivo is scarce or indirect. In the intact porcine heart, we investigated whether angiotensin II increases norepinephrine concentrations in the myocardial interstitial fluid (NEMIF) under basal conditions and during sympathetic activation and whether it enhances exocytotic and nonexocytotic ischemia-induced norepinephrine release. In 27 anesthetized pigs, NEMIF was measured in the left ventricular myocardium using the microdialysis technique. Local infusion of angiotensin II into the left anterior descending coronary artery (LAD) at consecutive rates of 0.05, 0.5, and 5 ng/kg per minute did not affect NEMIF, LAD flow, left ventricular dP/dtmax, and arterial pressure despite large increments in coronary arterial and venous angiotensin II concentrations. In the presence of neuronal reuptake inhibition and &agr;-adrenergic receptor blockade, left stellate ganglion stimulation increased NEMIF from 2.7±0.3 to 7.3±1.2 before, and from 2.3±0.4 to 6.9±1.3 nmol/L during, infusion of 0.5 ng/kg per minute angiotensin II. Sixty minutes of 70% LAD flow reduction caused a progressive increase in NEMIF from 0.9±0.1 to 16±6 nmol/L, which was not enhanced by concomitant infusion of 0.5 ng/kg per minute angiotensin II. In conclusion, we did not observe any facilitation of cardiac norepinephrine release by angiotensin II under basal conditions and during either physiological (ganglion stimulation) or pathophysiological (acute ischemia) sympathetic activation. Hence, angiotensin II is not a local mediator of cardiac sympathetic activity in the in vivo porcine heart.

In vivo evidence that EMD 57033 restores myocardial responsiveness to intracoronary Ca2+ in stunned myocardium

Despite ample in vitro evidence that myofilament Ca(2+)-responsiveness of stunned myocardium is decreased, in vivo data are inconclusive. Conversely, while Ca(2+)-sensitizing agents increase myofilament Ca(2+)-responsiveness in vitro, it has been questioned whether this also occurs in vivo. We therefore tested in open-chest anesthetized pigs whether EMD 57033 (the (+) enantiomer of 5-[1-(3,4-dimethoxybenzoyl)-1,2,3, 4-tetrahydro-6-quinolyl]-6-methyl-3,6-dihydro-2H-1,3, 4-thiadiazin-2-one) increases responsiveness to Ca(2+) of non-stunned myocardium and restores function of stunned myocardium by normalizing the responsiveness to Ca(2+). Studies were performed under beta-adrenoceptor blockade to minimize the contribution of the phosphodiesterase-III inhibitory actions of EMD 57033. Consecutive intracoronary Ca(2+) infusions were used to evaluate the contractile response (assessed by the left ventricular end-systolic elastance, E(es)) to added Ca(2+) of non-stunned myocardium and myocardium stunned by 15 min coronary artery occlusion and 30 min reperfusion. In non-stunned propranolol-treated myocardium, the Ca(2+) infusions doubled E(es) (baseline 6.9+/-0.9 mmHg mm(-2), n=8). Following Ca(2+)-washout, subsequent EMD 57033 infusion (0.1 mg kg(-1) min(-1), i.v.) tripled E(es) (P<0.05) and potentiated the Ca(2+)-induced increase in E(es) to 55.7+/-10.0 mmHg mm(-2) (P<0.05). Stunning (n=7) decreased E(es) to 5.3+/-0.6 mmHg mm(-2) (P>0.10) and attenuated the Ca(2+)-induced increase in E(es) (P<0.05). Subsequent infusion of EMD 57033 increased E(es) to 6.8+/-1.8 mmHg mm(-2) (P<0. 05) and restored responsiveness to added Ca(2+). These in vivo findings are consistent with the in vitro observations that myofilament Ca(2+)-responsiveness of stunned myocardium is reduced and that EMD 57033 increases contractility by enhancing myofilament Ca(2+)-responsiveness.