Spatial heterogeneity of myocardial perfusion predicts local potassium channel expression and action potential duration

AIMS: In the heart, there is not only a transmural gradient of left ventricular perfusion and action potential duration (APD), but also spatial heterogeneity within each myocardial layer, where local blood flow and energy turnover vary more than three-fold between individual regions. We analysed at high spatial resolution whether a corresponding heterogeneity also extends to ion channel gene expression and APD. METHODS AND RESULTS: In the open-chest beagle dog, left ventricular 300 microL samples of very low or high flow were identified by radioactive microspheres and expression levels determined by quantitative PCR. The distribution of epicardial APD was assessed by mapping local activation repolarization intervals (ARIs) and QT interval (QT). ERG, the potassium channel mediating IKr, and KChIP2, the interacting protein modulating Ito, were increased in Low flow (3.3- and 2.5-fold, P < 0.001 and <0.05, respectively; n = 6 hearts, 30-31 samples each) as compared with High flow areas. This suggested enhanced repolarizing currents in Low flow areas, and in consequence, mathematical model analysis predicted a shorter local APD upon enhanced ERG and IKr. Epicardial mapping revealed a patchy, temporally stable APD pattern (n = 11), a small apico-basal gradient and an APD prolongation induced by the ERG blocker dofetilide predominantly in areas of short basal ARI or QT, respectively (n = 9). In addition, in Short QT areas, ERG expression was three-fold increased (P < 0.05, n = 4). CONCLUSION: The spatial pattern of perfusion is matched by the novel patterns of K+ channel expression and APD. Whenever this newly recognized intramural dispersion of APD increases, it may contribute to arrhythmogenesis.     

Targeted disruption of cd73/ecto-5'-nucleotidase alters thromboregulation and augments vascular inflammatory response

To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5'-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (-14%; P<0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl3) was significantly reduced by 20% in cd73-/- mice (P<0.05). Bleeding time after tail tip resection tended to be shorter in cd73-/- mice (-35%). In vivo platelet cAMP levels were 0.96+/-0.46 in WT versus 0.68+/-0.27 pmol/106 cells in cd73-/- mice (P<0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 micromol/L) was undistinguishable between the two strains. In the cremaster model of ischemia-reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73-/- compared with WT littermates (WT 98% versus cd73-/- 245%; P<0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73-/- mice (P<0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo.     

A hemodynamic response to intravenous adenovirus vector particles is caused by systemic Kupffer cell-mediated activation of endothelial cells

Intravascular injection of adenoviral vectors may result in a toxic and potentially lethal reaction, the mechanism of which is poorly understood. We noted that mice demonstrated a transient change in behavior that was characterized by inactivity and lethargy within minutes after intravenous injection of relatively low doses of adenoviral vectors (including high-capacity gutless vectors). Moreover, immediately after vector injection a significant drop in blood pressure was measured that most probably was caused by the systemic activation of endothelial cells as monitored by detection of phosphorylated Akt/PKB kinase, activated endothelial nitric oxide synthase (eNOS), and nitrotyrosine. The activation of the endothelium was the result of the interaction of viral particles with Kupffer cells, which are resident macrophages of the liver representing the first line of defense of the innate immune system. Surprisingly, the uptake of vector particles by Kupffer cells not only resulted in their strong activation, but also in their nearly complete disappearance from the liver. Our results suggest that the toxicity of intravenously injected adenoviral vectors may be directly linked to the activation and destruction of Kupffer cells.     

Effects of Ketamine and Its Isomers on Ischemic Preconditioning in the Isolated Rat Heart

Background: Ischemic preconditioning protects the heart against subsequent ischemia. Opening of the adenosine triphosphate-sensitive potassium (KATP) channel is a key mechanism of preconditioning. Ketamine blocks KATP channels of isolated cardiomyocytes. The authors investigated the effects of ketamine and its stereoisomers on preconditioning.

Methods: Isolated rat hearts (n = 80) underwent 30 min of no-flow ischemia and 60 min of reperfusion. Two groups with eight hearts each underwent the protocol without intervention (control-1 and control-2), and, in eight hearts, preconditioning was elicited by two 5-min periods of ischemia before the 30 min ischemia. In the six treatment groups (each n = 8), ketamine, R (-)- or S (+)-ketamine were administered at concentrations of 2 or 20 [mu]g/ml before preconditioning. Eight hearts received 20 [mu]g/ml R (-)-ketamine before ischemia. Left ventricular (LV) developed pressure and creatine kinase (CK) release during reperfusion were determined as variables of ventricular function and cellular injury.

Results: Baseline LV developed pressure was similar in all groups: 104 +/- 28 mmHg (mean +/- SD). Controls showed a poor recovery of LV developed pressure (17 +/- 8% of baseline) and a high CK release (70 +/- 17 IU/g). Ischemic preconditioning improved recovery of LV developed pressure (46 +/- 14%) and reduced CK release (47 +/- 17 IU/g, both P < 0.05 vs. control-1). Ketamine (2 [mu]g/ml) and 2 or 20 [mu]g/ml S (+)-ketamine had no influence on recovery of LV developed pressure compared with preconditioning (47 +/- 18, 43 +/- 8, 49 +/- 36%) and CK release (39 +/- 8, 30 +/- 14, 41 +/- 25 IU/g). After administration of 20 [mu]g/ml ketamine and 2 or 20 [mu]g/ml R (-)-ketamine, the protective effects of preconditioning were abolished (LV developed pressure-recovery, 16 +/- 14, 22 +/- 21, 18 +/- 11%; CK release, 67 +/- 11, 80 +/- 21, 82 +/- 41 IU/g; each P < 0.05 vs. preconditioning). Preischemic treatment with R (-)-ketamine had no effect on CK release (74 +/- 8 vs. 69 +/- 9 IU/g in control-2, P = 0.6) and functional recovery (LV developed pressure 12 +/- 4 vs. 9 +/- 2 mmHg in control-2, P = 0.5).     

Direct comparison of magnetic resonance imaging and conductance microcatheter in the evaluation of left ventricular function in mice

The aim of the present work was to study the reliability of conductance microcatheter volumetric measurements as compared to magnetic resonance imaging (MRI) in the same set of mice. Mice left ventricular (LV) volumes were monitored under basal conditions and in a hypertrophy model induced by transverse aortic constriction (TAC). Cardiac function was evaluated in isoflurane anesthetized mice (n = 8) by MRI followed by 1.4 F Millar microtip catheter measurements. The second group of mice with TACinduced cardiac hypertrophy was studied eight weeks after surgery. Reliability of 3D–reconstructed MRI data was confirmed by comparison with autopsy masses (autopsy LV mass = 73.6 ± 3.4 mg; MRI LV mass = 76.9 ± 3.7 mg). Conduction catheter was found to greatly underestimate end–diastolic and end–systolic volumes and thus stroke volume as well as cardiac output in control mice (MRI: EDV = 79 ± 8 μl, ESV = 27±9 μl, SV = 51 ± 9 μl, CO = 25 ± 6 ml/min; Catheter: EDV = 28 ± 5 μl, ESV = 8 ± 4 μl, SV = 19 ± 4 μl, CO = 10 ± 2 ml/min). However, values for ejection fraction showed no significant differences between the two methods. In the hypertrophy model, stroke volume and cardiac output were increased when measured with MRI (SV: +19 ± 20%; CO: +28 ± 27%), whereas catheter data showed opposite directional changes (SV: –22 ± 37%; CO: –31 ± 37%). Ejection fraction was found to be reduced only in catheter measurements (–31 ± 26%). In summary, our data demonstrate that absolute volumetric values are strikingly underestimated by conduction catheter measurements and that even detection of directional changes with this method may not always be feasible. Drs. Jacoby and Molojavyi contributed equally to this work.     

Thiopentone does not block ischemic preconditioning in the isolated rat heart

PURPOSE: Ischemic preconditioning protects the heart against subsequent prolonged ischemia by opening of adenosine triphosphate-sensitive potassium (K(ATP)) channels. Thiopentone blocks K(ATP) channels in isolated cells. Therefore, we investigated the effects of thiopentone on ischemic preconditioning. METHODS: Isolated rat hearts (n=56) were subjected to 30 min of global no-flow ischemia, followed by 60 min of reperfusion. Thirteen hearts underwent the protocol without intervention (control, CON) and in 11 hearts (preconditioning, PC), ischemic preconditioning was elicited by two five-minute periods of ischemia. In three additional groups, hearts received 1 (Thio 1, n=11), 10 (Thio 10, n=11) or 100 microg x mL(-1) (Thio 100, n=10) thiopentone for five minutes before preconditioning. Left ventricular (LV) developed pressure and creatine kinase (CK) release were measured as variables of myocardial performance and cellular injury, respectively. RESULTS: Recovery of LV developed pressure was improved by ischemic preconditioning (after 60 min of reperfusion, mean +/- SD: PC, 40 +/- 19% of baseline) compared with the control group (5 +/- 6%, P <0.01) and this improvement of myocardial function was not altered by administration of thiopentone (Thio 1, 37 +/- 15%; Thio 10, 36 +/- 16%; Thio 100, 38 +/- 16%, P=0.87-0.99 vs PC). Total CK release over 60 min of reperfusion was reduced by preconditioning (PC, 202 +/- 82 U x g(-1) dry weight) compared with controls (CON, 383 +/- 147 U x g(-1), P <0.01) and this reduction was not affected by thiopentone (Thio 1, 213 +/- 69 U x g(-1); Thio 10, 211 +/- 98 U x g(-1); Thio 100, 258 +/- 128 U x g(-1), P=0.62-1.0 vs PC). CONCLUSION: These results indicate that thiopentone does not block the cardioprotective effects of ischemic preconditioning in an isolated rat heart preparation.     

Effects of ketamine and its isomers on ischemic preconditioning in the isolated rat heart

BACKGROUND: Ischemic preconditioning protects the heart against subsequent ischemia. Opening of the adenosine triphosphate-sensitive potassium (KATP) channel is a key mechanism of preconditioning. Ketamine blocks KATP channels of isolated cardiomyocytes. The authors investigated the effects of ketamine and its stereoisomers on preconditioning. METHODS: Isolated rat hearts (n = 80) underwent 30 min of no-flow ischemia and 60 min of reperfusion. Two groups with eight hearts each underwent the protocol without intervention (control-1 and control-2), and, in eight hearts, preconditioning was elicited by two 5-min periods of ischemia before the 30 min ischemia. In the six treatment groups (each n = 8), ketamine, R(-)- or S(+)-ketamine were administered at concentrations of 2 or 20 microg/ml before preconditioning. Eight hearts received 20 microg/ml R(-)-ketamine before ischemia. Left ventricular (LV) developed pressure and creatine kinase (CK) release during reperfusion were determined as variables of ventricular function and cellular injury. RESULTS: Baseline LV developed pressure was similar in all groups: 104 +/- 28 mmHg (mean +/- SD). Controls showed a poor recovery of LV developed pressure (17 +/- 8% of baseline) and a high CK release (70 +/- 17 IU/g). Ischemic preconditioning improved recovery of LV developed pressure (46 +/- 14%) and reduced CK release (47 +/- 17 IU/g, both P < 0.05 vs. control-1). Ketamine (2 microg/ml) and 2 or 20 microg/ml S(+)-ketamine had no influence on recovery of LV developed pressure compared with preconditioning (47 +/- 18, 43 +/- 8, 49 +/- 36%) and CK release (39 +/- 8, 30 +/- 14, 41 +/- 25 IU/g). After administration of 20 microg/ml ketamine and 2 or 20 microg/ml R(-)-ketamine, the protective effects of preconditioning were abolished (LV developed pressure-recovery, 16 +/- 14, 22 +/- 21, 18 +/- 11%; CK release, 67 +/- 11, 80 +/- 21, 82 +/- 41 IU/g; each P < 0.05 vs. preconditioning). Preischemic treatment with R(-)-ketamine had no effect on CK release (74 +/- 8 vs. 69 +/- 9 IU/g in control-2, P = 0.6) and functional recovery (LV developed pressure 12 +/- 4 vs. 9 +/- 2 mmHg in control-2, P = 0.5). CONCLUSION: Ketamine can block the cardioprotective effects of ischemic preconditioning. This effect is caused by the R(-)-isomer.