Heart rate adjustment of magnetic field map rotation in detection of myocardial ischemia in exercise magnetocardiography

Aims We studied the capability of heart rate (HR) adjusted change in multichannel magnetocardiogram (MCG) to detect exercise-induced ischemia. Methods and results The MCG and 12-lead ECG were recorded simultaneously during supine exercise testing in 17 healthy controls and 24 patients with single vessel coronary artery disease (CAD). In the MCG analysis, we plotted the orientation of the magnetic field map (MFM) against the HR in each cardiac cycle during recovery. A regression line was fitted to the data and the line slope (degrees/bpm) was determined. In the ECG, the ST-segment depression vs HR (ST/HR) slope was evaluated. The HR adjusted MFM rotation was more extensive in the pooled CAD group, and in all subgroups with different stenosed vessel, than in the control group at the ST-segment (1.5 ± 2.1°/bpm vs 0.29 ± 0.25°/bpm, p < 0.0005) and at the T-wave apex (0.95 ± 0.81°/bpm vs 0.24 ± 0.25°/bpm, p < 0.0005). Areas under the receiver operating characteristic curves of the HR adjusted MFM rotation at the ST-segment (88.5 %) and the T-wave (86.0 %) were higher than the ones without HR adjustment (75.5 % and 68.1 %, respectively), and higher than the area of ST/HR slope in the ECG (80.2 %). Conclusion HR adjusted MFM rotation detects transient ischemia independent of the stenosed vessel. HR adjustment improves the performance of the MCG in ischemia detection by the analysis of the ST-segment and the T-wave. The MCG was superior to the 12-lead ECG. Received: 5 April 2001, Returned for 1. revision: 7 May 2001, 1. Revision received: 25 May 2001, Returned for 2. revision: 12 June 2001, 2. Revision received: 18 June 2001, Accepted: 20 June 2001     

Effect of atrial dilatation on electrophysiologic properties and inducibility of atrial fibrillation

Introduction: Atrial dilatation may play an important role in the occurrence of atrial fibrillation (AF) in clinical situations. However, the electrophysiologic characteristics of dilated atria are still unclear. Methods and results: In 18 isolated Langendorff-perfused canine hearts (14.6 ± 2.2 kg), we measured atrial effective refractory periods (ERPs) at four different sites, conduction velocity and percentage of slow conduction on the right atrium (using a high-density electrode plaque), and assessed the inducibility of AF at the baseline (0 cm H₂O) and high (15 cm H₂O) atrial pressure. The atrial ERPs did not change significantly, but the dispersion of ERP increased significantly (40 ± 18 vs 25 ± 9 vs ms, p = 0.01) during high atrial pressure. The percentage of slow conduction (< 25 cm/s) over the mapping area, and the inducibility of AF increased during high atrial pressure (23.7 ± 10.2 % vs 32.1 ± 12.5 %, p = 0.02). The AF inducibility significantly correlated with the ERP dispersion (R = 0.75, p < 0.001) and maximal percentage of slow conduction (R = 0.88, p < 0.001). Furthermore, ERPs were significantly shorter in the induced AF group than those without induced AF (68 ± 17 vs 84 ± 16 ms, P < 0.05). Conclusions: The increased inhomogenity in atrial electrophysiological properties during atrial dilatation contributed to the inducibility of AF. Received: 26 November 2001?Returned for 1. revision: 2 January 2002?1. Revision received: 11 February 2002?Returned for 2. revision: 25 March 2002?2. Revision received: 6 May 2002?Returned for 3. revision: 10 June 2002?3. Revision received: 21 August 2002?Accepted: 11 September 2002     

Inhibition of apoptotic responses after ischemic stress in isolated hearts and cardiomyocytes

Recent findings on the induction of anti-apoptotic gene expression in ischemic/reperfused hearts encouraged us to investigate whether ischemic/reperfused hearts may be protected against apoptosis induction. To analyze this hypothesis we performed studies on isolated perfused hearts of rat. For apoptosis induction, hearts were perfused with the NO donor (±)-S-nitroso-N-acetylpenicillamine (SNAP, 10 μM) for 30 minutes. Four hours thereafter apoptosis was detected by DNA laddering and TUNEL assay. Under normoperfusion SNAP induced 5.5 ± 1.4 TUNEL-positive myocytes per tissue section (vs. 1.8 ± 0.5 in controls). But when hearts were subjected to 20 minutes of no flow ischemia, which was sufficient for energy depletion of the hearts without inducing severe necrotic or apoptotic cell death, reperfusion in the presence of SNAP did not induce apoptosis. To analyze if this mode of protection is a property of the cardiomyocytes, we performed corresponding experiments on ventricular cardiomyocytes of rat. Again, under normoxic conditions SNAP (100 (μM) increased the number of TUNEL-positive cells to 12.6 ± 4.9 % (vs. 5.4 ± 0.7 % in controls). But when SNAP was added after 3 h of simulated ischemia, which was sufficient for energy depletion of the cells without inducing apoptotic cell death, the number of apoptotic cells did not increase. The ischemia-induced protection of hearts and cardiomyocytes goes along with an increased expression of several anti-apoptotic genes, mainly of the bcl-2 family. This indicates that ischemic conditions induce an anti-apoptotic gene program in cardiomyocytes, which may also be responsible for the observed anti-apoptotic actions in the intact ischemic/reperfused myocardium. Received: 20 March 2002, Returned for 1. revision: 8 April 2002, 1. Revision received: 30 April 2002, Returned for 2. revision: 21 May 2002, 2. Revision received: 29 May 2002, Returned for 3. revision: 29 May 2002, 3. Revision received: 6 June 2002, Accepted: 12 June 2002 Correspondence to: Dr. G. Taimor     

Induction of atrial fibrillation in mice by rapid transesophageal atrial pacing

Objective: Atrial fibrillation (AF) as an “indicator arrhythmia” for enhanced atrial vulnerability in mouse hearts has not yet been systematically examined. We therefore evaluated a transesophageal rapid atrial stimulation protocol for the induction of AF in C57Bl/6 mice. Methods: 40 C57Bl/6 mice (19 female and 21 male; 5.2 ± 2.1 months; 18 – 27 g) were examined by closed chest transesophageal atrial stimulation. Baseline ECG and electrophysiological parameters, AF-inducing stimulation cycle length (CL) and AF duration were analyzed. Results: The surface ECG demonstrated a significantly faster heart rate in female mice (R-R: 138.7 ± 19.9 ms versus 150.5 ± 15.7 ms, P < 0.05). AF was inducible in 90 % of the population and not inducible in 4 mice, all female (21 % in this subgroup). Mean induction CL was 27.4 ± 7.3 ms. Mean AF duration was 26.9 ± 42.6 s before spontaneous termination. In a subgroup of 4 female and 4 male mice (mean age 7.5 months), successive testing of AF induction showed a range of higher susceptibility to AF at stimulus amplitudes of 3.0 – 4.0 mA and stimulation CLs between 15 – 25 ms. AF induction was observed to be constantly reproducible in the individual animals. No correlation to pacing stimulus length and amplitude was found. Conclusions: This study demonstrates that it is possible to reproducibly induce self-terminating AF and supraventricular arrhythmias in mice by transesophageal atrial burst stimulation. The presented method allowing serial testings of the same animal can be a useful tool in further investigations with transgenic mice and might be helpful in the characterization of underlying genetic or molecular mechanisms of AF. Received: 26 April 2002, Returned for revision: 21 May 2002, Revision received: 17 June 2002, Accepted: 24 June 2002 Correspondence to: J. W. Schrickel, MD     

Magnetocardiographic QT dispersion during cardiovascular autonomic function tests

QT dispersion is considered to reflect nonhomogeneity of ventricular repolarization. The autonomic nervous system modulates QT interval duration, but the effect may not be spatially homogenous. Magnetocardiography (MCG) registers the weak magnetik fields generated by myocardial electric currents with high localizing accuracy. We studied the effect of rapid cardiovascular autonomic nervous adjustment on QT dispersion in MCG. Ten healthy male volunteers were monitored during deep breathing, the Valsalva maneuver, sustained handgrip, hyperventilation, the cold pressor test and mental stress. 67 MCG channels and 12 ECG leads were recorded simultaneously. A computer algorithm was used for QT interval measurements. QT dispersion was defined as maximum – minimum or standard deviation of the QT_peak and QT_end intervals. In MCG the QT_end dispersion increased during deep inspiration compared with deep expiration (96±19 ms v 73±27 ms, p=0.05). Magnetic QT dispersion tended to increase during the bradycardia phase of the Valsalva maneuver, but the change was obvious only for QT_end (55±26 ms v 76±29 ms, p<0.05) Other tests had no significant effect on QT dispersion, not even the cold pressor test, although it causes strong sympathetic activation. Magnetic and electric QT_peak and QT_end intervals correlated closely (r=0.93 and 0.91), whereas the QT dispersion measures showed no correlation. In conclusion, magnetic QT dispersion is not modified by rapid changes in autonomic tone, but maneuvers involving deep respiratory efforts and changes in ventricular loading affect QT dispersion measurements. Received: 4 April 2000 Returned for revision: 2 May 2000 Revision received: 20 June 2000 Accepted: 10 July 2000     

Endogenous nitric oxide regulates right coronary blood flow during acute pulmonary hypertension in conscious dogs

This study investigated the role of nitric oxide (NO) in the control of right coronary (RC) blood flow at rest and during acute pulmonary hypertension. Experiments were performed in seven chronically instrumented, conscious dogs. NO synthesis was inhibited by systemic administration of N^ω-nitro-L-arginine (LNA, 35 mg/kg). Inflation of a balloon in the main pulmonary artery raised right ventricular (RV) peak systolic pressure from 34 ± 2 to 47 ± 3 mmHg before LNA and from 37 ± 2 to 47 ± 3 mmHg after LNA, but did not affect mean systemic arterial pressure. RV O₂ consumption (MVO₂) increased from 4.4 ± 0.7 to 6.1 ± 0.7 ml/min/100 g. 82 % of the elevated RV MVO₂ was provided by RC blood flow, which increased from 46 ± 7 to 61 ± 8 ml/min/100 g. After LNA, resting RV MVO₂ and RC flow fell. RC venous PO₂ fell, but RV lactate uptake was not altered. During pulmonary hypertension, the increase in RC blood flow was blunted by LNA, so that only 66 % of the elevated RV MVO₂ was supplied by increased RC flow. Analysis of O₂ supply variables as functions of RV MVO₂ further demonstrated a significant role of NO in regulating RC flow at rest and during moderate pulmonary hypertension. Conclusions NO is required for the RC hyperemic response to acute pulmonary hypertension as well as for normal resting RC blood flow. After blockade of NO synthesis, RV O₂ supply at rest and during pulmonary hypertension was sustained by increased RV O₂ extraction. Received: 2 April 2002, Returned for 1. revision: 17 April 2002, 1. Revision received: 13 May 2002, Returned for 2. revision: 29 May 2002, 2. Revision received: 5 June 2002, Accepted: 10 June 2002     

Recording locations in multichannel magnetocardiography and body surface potential mapping sensitive for regional exercise-induced myocardial ischemia

Introduction This study aimed to identify the optimal locations in multichannel magnetocardiography (MCG) and body surface potential mapping (BSPM) to detect exercise-induced myocardial ischemia. Methods We studied 17 healthy controls and 24 coronary artery disease (CAD) patients with stenosis in one of the main coronary artery branches: left anterior descending (LAD) in 11 patients, right (RCA) in 7 patients, and left circumflex (LCX) in 6 patients. MCG and BSPM signals were recorded during a supine bicycle stress test. The capability of a recording location to separate the groups was quantified by subtracting the mean signal amplitude of the normal group from that of the patient group during the ST segment and at the T-wave apex, and dividing the resulting amplitude difference by the corresponding standard deviation within all subjects. Results In MCG the optimal location for ST depression was at the right inferior grid for the RCA, at the mid-inferior grid for the LCX, and in the middle of these locations for the LAD subgroup (mean ST amplitudes: CAD −80 ± 360fT, controls 610 ± 660fT; p < 0.001). In BSPM it was on the left upper anterior thorax for the LAD, left lower anterior thorax for the RCA, and on the lower back for the LCX subgroup (mean ST amplitudes: CAD −39 ± 61 μV and controls 38 ± 38 μV; p < 0.001). In MCG the optimal site for T-wave amplitude decrease was the same as the one for the ST depression. In BSPM it was on the middle front for the LAD, on the back for the LCX and on the left abdominal area for the RCA group. In accordance with electromagnetic theory, the largest ST segment and T-wave amplitude changes took place in MCG in locations orthogonal to those in BSPM. Conclusion This study identified magnetocardiographic and BSPM recording locations which are sensitive for detecting transient myocardial ischemia by evaluation of the ST segment as well as the T-wave. These locations strongly depend on ischemic regions and are outside the conventional 12-lead ECG recording sites. Received: 9 October 2000 / Returned for revision: 23 October 2000 / Revision received: 2 January 2001 / Accepted: 19 January 2001     

The expression of heat shock protein 60 in myocardium of patients with chronic atrial fibrillation

Objective Cardiomyocytes respond to stress with the expression of different heat shock proteins (HSP). HSP60 is induced by various stress factors. The aim of this study was to investigate the expression of HSP60 in human atrial fibrillation (AF). Method Right atrial samples from 14 patients undergoing elective cardiac surgery were excised and immediately frozen in liquid nitrogen. Eight patients had chronic AF and six patients were in sinus rhythm. The HSP60 protein level was determined by SDS-PAGE, Western blot and quantified by optical densitometry according to the immunoreactive bands of actin. Results In myocardial samples from patients with chronic AF, we found a more than 2.5-fold increase in HSP60 expression compared to atrial myocardium of patients in sinus rhythm. Conclusion This result indicates an up regulation of HSP60 in response to chronic atrial fibrillation Received: 31 October 2001, Returned for 1. revision: 20 Novemver 2001, 1. Revision received: 12 December 2001, Returned for 2. revision: 3 January 2002, 2. Revision received: 25 January 2002, Accepted: 6 February 2002     

Clofilium in the isolated perfused rabbit heart: A new model to study proarrhythmia induced by class III antiarrhythmic drugs

Objective: The clinical usefulness of class III antiarrhythmic drugs for the treatment of tachyarrhythmias is limited by their potential proarrhythmic effects, mainly torsades-depointes (TdP). The goal of this experimental study was to develop an isolated whole-heart model exhibiting typical characteristics of class III drug-induced ventricular arrhythmias. Methods: Isolated rabbit hearts were perfused with a Krebs-Henseleit buffer containing 10 μM clofilium and then exposed to a modified Krebs-Henseleit buffer with 2.0 mM K² and 0.5 mM Mg²⁺. Hearts subjected to either clofilium alone or modified buffer alone were used as controls. Results: Under clofilium the QT interval increased from 187±16 to 282±33 ms. Within 8 to 25 s after the change of the perfusate, ventricular arrhythmias developed in all hearts associated with a further QT prolongation to 380±73 ms when the first ventricular extrasystole occurred. Simultaneously, the monophasic action potential durations increased relatively more during late repolarization; from 99±21 to 110±25 ms (+11%) at 50% repolarization, from 143±24 to 178±40 ms (+24%) at 70%, and from 200±30 to 275±53 ms (+38%) at 90%. The predominant rhythm was polymorphic with either two alternating or multiple QRS morphologies exhibiting the characteristic features of torsades-depointes. All control hearts stayed in normal sinus rhythm. Conclusion: Under the conditions selected, the isolated perfused rabbit heart represents a useful experimental approach to study the proarrhythmic effects of class III agents. This model provides a convenient way to manipulate the ionic and pharmacologic millieu in a preparation conserving the functional anatomy of the whole organ without interference by cardiovascular reflexes. It might be useful for analyzing the conditions favoring and preventing drug-induced torsades-depointes. Received: 13 August 1997, Returned for revision: 10 September 1997, Revision received: 29 September 1997, Accepted: 22 October 1997     

Cyclic GMP attenuates cyclic AMP-stimulate inotropy and oxygen consumption in control and hypertrophic hearts

We tested the hypothesis that increasing myocardial cyclic GMP would attenuate cyclic AMP induced positive inotropy and O₂ consumption, in part, through changes in cyclic AMP and that renal hypertension-induced cardiac hypertrophy (HYP) would alter this relationship. Anesthetized, open chest rabbits (N = 48) were divided into four groups of control (CON) and HYP animals which received vehicle (VEH), isoproterenol 10⁻⁶M (ISO), 3-morpholinosyndnonimine 10⁻⁴M, (SIN-1), or a combination of ISO+SIN-1. Coronary blood flow (micro-spheres) and O₂ extraction (microspectrophotometry) were used to determine O₂ consumption in both subepicardium (EPI) and subendocardium (ENDO). Left ventricular change in wall thickness (%) was increased significantly by ISO in both CON (16 ± 4 to 31 ± 6) and HYP (17 ± 2 to 24 ±3). Percent change in wall thickness was similar in the CON, SIN-1, and ISO+SIN-1 groups. Myocardial O₂ consumption (ml O₂/min/100 g) was increased by ISO in CON (10.3 ± 1.0 to 13.6 ± 2.0 EPI; 10.9 ± 1.0 17.1 ±1.7 ENDO) and HYP (8.2 ± 1.4 to 12.3 ± 2.2 EPI; 6.6 ± 1.4 to 14.8 ± 1.8 ENDO). Oxygen consumption was unaffected by SIN-1 in CON and HYP animals. ISO+SIN-1 caused attenuated ISO-induced increases in O₂ consumption in CON in EPI and ENDO, and in EPI in HYP. Cyclic GMP (pmol/g) was unchanged by ISO in CON and HYP, and increased by SIN-1 in CON (8.1 ± 1.3 to 19.2 ± 2.3 EPI) and HYP (9.1 ± 1.5 to 12.8 ± 2.0 EPI). Cyclic GMP remained elevated with ISO+SIN-1 in both groups. Cyclic AMP (pmol/g) was increased significantly by ISO in CON (496 ± 43 to 725 ± 106 EPI; 534 ± 44 to 756 ± 148 ENDO) and insignificantly in HYP (435 ± 50 to 566 ± 35 EPI; 497 ± 51 to 583 ± 47 ENDO). Cyclic AMP levels were unaffected by SIN-1 in either group. Isoproterenol induced increases in cyclic AMP were blunted by ISO+SIN-1 in CON (496 ± 43 to 537 ± 59 EPI) and not affected in HYP. The current study demonstrated attenuation of cyclic AMP mediated increased inotropy and O₂ consumption by increasing cyclic GMP, which appeared, in part, related to cyclic GMP-induced reduction in cyclic AMP. This effect of cyclic GMP on cyclic AMP was not observed in myocardial hypertrophy. Received: 4 January 1999, Returned for 1. revision: 29 January 1999, 1. Revision received: 30 March 1999, Returned for 2. Revision: 3 May 1999, 2. Revision received: 3 May 1999, Returned for 3. Revision: 12 May 1999, 3. Revision received: 23 June 1999, Returned for final revision: 7 July 1999, Accepted: 22 July 1999     

Impaired coronary flow and left ventricular dysfunction after mechanical recanalization in acute myocardial infarction: role of neurohumoral activation?

Background: Reopening of the infarct-related coronary artery is the treatment of choice in the clinical setting of acute myocardial infarction. Nevertheless the removal of the total occlusion obtained either by thrombolysis or by primary angioplasty is followed by the ischemia/reperfusion sequelae. One of many proposed mechanisms playing a role in ischemia/reperfusion damage is a persistent increase in vasoconstrictor tone, which reduces cardiac function and impairs myocardial blood flow during primary percutaneous coronary intervention in acute myocardial infarction (PAMI). Methods: To investigate early neurohumoral changes during PAMI we enrolled 18 patients, who were collated to 13 patients with stable angina undergoing elective PTCA. To evaluate angiotensin II (AngII), endothelin-1 (ET-1), vasopressin (AVP), norepinephrine (NE), troponin T (TNT), creatinephosphate kinase (CPKM) and isoenzyme MB (CPKMB), we collected blood from the pulmonary artery before and immediately after the infarct-related artery (IRA; TIMI 0 → 2–3) or culprit lesion revascularization. Hemodynamic and angiographic LV-function parameters were compared to biochemical data. Corrected TIMI-frame count (CTFC) was used as an index of coronary blood flow and correlated to the biochemical measurements. Results: CTFC in the IRA correlated inversely (p = 0.03; r = −0.51) with left ventricular ejection fraction measured after 10 days, and positively (p = 0.03; r = 0.54) with the maximal amount of LDH released after onset of AMI. There was an abrupt and long lasting rise in ET-1 (+65 %; p < 0.001) and an instant short lasting increase in AVP (+37 %; p < 0.05), whereas NE concentrations were elevated prior to PAMI and remained elevated during reperfusion. Correlations with CTFC were found for ET-1 (p = 0.01; r = 0.61) and NE (p = 0.01; r = 0.58) during reperfusion. The extent of left ventricular dysfunction correlated with the concentrations of AVP and NE during reperfusion. Conclusions: There is evidence for a distinct pattern of neurohumoral activation during early reperfusion in acute myocardial infarction. In particular, we documented substantial increases in AVP and ET-1. Left ventricular wall-stress appears to be involved in the release of AVP. Elevated levels of ET-1 and NE are associated with impaired angiographic reperfusion and increased myocardial damage after mechanical recanalization. Received: 13 December 2001, Returned for 1. revision: 14 January 2002, 1. Revision received: 10 April 2002, Returned for 2. revision: 30 April 2002, 2. Revision received: 10 May 2002, Accepted: 13 May 2002     

Expression of voltage-gated K<Superscript>+</Superscript> channels in human atrium

Voltage-gated K⁺ channels underlie repolarisation of the cardiac action potential and represent a potential therapeutic target in the treatment of cardiac dysrhythmias. However, very little is known about the relative expression of K⁺ channel subunits in the human myocardium. We used a semi-quantitative RT-PCR technique to examine the relative expression of mRNAs for the voltage-gated K⁺ channel subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, KvLQT1, HERG and IsK in samples of human atrial appendage. Data were expressed as a percentage expression density relative to an 18S ribosomal RNA internal standard. The most abundant K⁺ channel mRNAs were Kv4.3 (80.7 ± 10.1 %), Kv1.5 (69.7 ± 11.2 %) and HERG (55.9 ± 21.5 %). Significant expression of KvLQT1 (33.5 ± 5.5 %,) and Kv1.4 (26.7 ± 9.6 %) was also detected. Levels of mRNAs for Kv1.2 and IsK were very low and neither Kv2.1 nor Kv4.2 mRNA were detected in any experiments. Whole-cell patch-clamp techniques were used to examine the outward currents of isolated human atrial myocytes at 37 °C. These recordings demonstrated the existence of transient (I_to1) and sustained (I_so) outward currents in isolated human atrial myocytes. I_to1, and not I_so, showed voltage-dependent inactivation during 100 ms pre-pulses. Both I_to1 and I_so were inhibited by high concentrations (2 mM) of the K⁺ channel blocker, 4-aminopyridine (4-AP). However, lower concentrations of 4-AP (10 μM) inhibited I_so selectively. I_to1 recovered from inactivation relatively rapidly (t ∼21 ms). These data, with published information regarding the properties of expressed K⁺ channels, suggest that Kv4.3 represents the predominant K⁺ channel subunit underlying I_to1 with little contribution of Kv1.4. The sensitivity of Iso to very low concentrations of 4-aminopyridine and the relatively low expression of mRNA for Kv1.2 and Kv2.1 is consistent with the major contribution of Kv1.5 to this current. The physiological significance of the expression of KvLQT1 and Kv1.4 mRNA in the human atrium warrants further investigation. Received: 30 August 2000, Returned for 1. revision: 21 September 2000, 1. Revision received: 21 June 2002, Returned for 2. revision: 15 July 2002, 2. Revision received: 30 July 2002, Accepted: 31 July 2002 Correspondence to: Dr. A. F. James     

Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo

Vectors based on lentiviruses such as human immunodeficiency virus (HIV) type-1 have many advantages for gene therapy, including the ability to infect non-dividing cells, long-term transgene expression and the absence of induction of an inflammatory/immune response. This study was initiated to determine whether lentiviruses would efficiently transfer genes to both neonatal and adult cardiac cells in culture and, by direct injection, to the heart in vivo. A three-plasmid expression system, including a packaging defective helper construct, a plasmid coding for a heterologous (VSV-G) envelope protein and a vector construct harboring reporter genes –E-GFP (enhanced green fluorescent protein) and puro (puromycin-resistance protein) was used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells. We demonstrated efficient gene transfer into neonatal and adult cardiac myocytes in vitro and identified conditions in which virtually 100 % of cultured neonatal and 70 % of adult cardiac myocytes express the reporter gene. Transduction of adult cardiac myocytes with high titre lentiviral vectors did not affect the cell number, morphology or viability compared to untransduced cells. We delivered HIV-1-based vectors to the intact heart by direct injection. Hearts transduced with pseudotyped HIV-1 vectors showed levels of transgene expression comparable to that achieved by adenovirus vectors. This study demonstrates for the first time that lentivirus-based vectors can successfully transduce adult cardiomyocytes both in vitro and in vivo, and opens up the prospect of lentivirus-based vectors becoming an important gene delivery system in the cardiovascular field. Received: 1 October 2001, Returned for 1. revision: 18 October 2001, 1. Revision received: 19 November 2001, Returned for 2. revision: 6 December 2001, 2. Revision received: 13 February 2002, Accepted: 6 March 2002     

Delayed attenuation of myocardial ischemia with repeated exercise in subjects with stable angina: a possible model for the second window of protection?

Aims: A delayed myocardial protection extends between 24 and 96 h after ischemic preconditioning in animals. To test this phenomenon in humans, subjects with stable angina were subjected to exercise test-induced myocardial ischemia and the effect of this “preconditioning” ischemic insult on the exercise-induced myocardial ischemia with the re-exercise after 24–96 hours was studied. Methods and results: Forty-eight males with a history of infarction and positive exercise test were recruited to the study. After baseline symptom-limited exercise test, the subjects were randomized to four experimental groups (n=12/group). The groups were allowed to recover for 24 h, 48 h, 72 h or 96 h before performing the second exercise test. Variables analyzed were heart rate-systolic blood pressure product at 1 mm ST segment depression, time to 1 mm ST segment depression, maximum ST segment depression, exercise duration, and the total ischemic time. There were no intergroup differences in baseline values for these variables. All variables were significantly improved at 24 h, the improvement peaked usually at 48 h (maximum increase in the variables by 31–46%), and the variables returned to baseline by 96 h after the first test. Conclusions: The exercise-induced ischemia caused transient attenuation of myocardial ischemia with re-exercise. Although the time-window and the time-course of this effect shows striking resemblance to those of the delayed preconditioning in animals, its mechanism remains speculative. The most probable mechanisms that may be involved include increased myocardial perfusion and/or some adaptive changes in the myocardium, the delayed preconditioning being one possibility. Received: 2 February 2000 Returned for 1. revision: 23 February 2000 1. Revision received: 3 April 2000 Returned for 2. revision: 3 May 2000 2. Revision received: 20 May 2000 Accepted: 26 May 2000     

Ventricular fibrillation during acute coronary occlusion is related to the dilation of the ischemic region

Myocardial stretch induces several electrophysiological changes and arrhythmias, but little is known on its possible role in triggering ventricular fibrillation (VF) during acute coronary occlusion. In thiopental-anesthetized, open-chest pigs submitted to a 40-min ligation of the left anterior descending coronary artery, the association between the early increase in end-diastolic length (measured by means of ultrasonic crystals) in the ischemic region and subsequent VF was analyzed. Animals received no treatment (n = 35) or intravenous nitroglycerin (2.5 μg/kg/min for 20 min, starting 10 min after coronary occlusion, n = 8) or Gd³⁺ (80 μM/kg for 35 min, starting 5 min before occlusion, n = 15). Twenty-four animals (41 %) had VF, 16 to 39 min after coronary occlusion. The magnitude of ischemic dilation and the incidence of VF were similar among groups. End-diastolic length in the ischemic region 15 min after coronary occlusion was 115.7 ± 1.2 % of baseline in animals with VF and 111.4 ± 0.9 % in those without (P = 0.007), and was the strongest predictor of this arrhythmia (P = 0.003) after adjusting for treatment and other possible confounding variables. Thus, the dilation of the ischemic region is closely and independently associated with VF following coronary occlusion. Although the interventions tested in the present study failed to protect against this arrhythmia, the results strongly suggest an influence of ischemic dilation on VF. Received: 11 April 2002, Returned for revision: 21 May 2002, Revision received: 12 June 2002, Accepted: 27 June 2002 Correspondence to: D. Garcia-Dorado, M.D.     

Reduction of oxygen delivery during post-ischemic reperfusion protects the isolated guinea pig heart

Objective: To further characterise the influence of oxygen delivery during early reperfusion (first 5 min) in the isolated guinea pig heart, three modes of coronary reperfusion were chosen, differing with respect to reperfusion flow and arterial PO₂. Methods: Isolated working guinea pig hearts underwent ischemia and reperfusion (15 min each). Reperfusion was at constant pressure (Group 1, 60 mmHg, n = 7) or at constant flow (Group 2, 5 ml/min, n = 7) with a PO₂ of 600 mmHg. Group 3 (n = 8) was reperfused at 5 ml/min with a PO₂ of 300 mmHg for 5 min and a PO₂ of 600 mmHg thereafter. Lactate release and oxygen consumption were determined during reperfusion. Glutathione release served to assess myocardial oxidative stress. Results: After ischemia, hearts in Group 1 (mean coronary flow 14.4±1.1 ml/min during the first 5 min of reperfusion) performed external heart work at 31 ± 2 % of the pre-ischemic level. Performance in Group 2 recovered to 50 ± 3 % and in Group 3 to 68 ± 3 %. Myocardial oxygen consumption during early reperfusion (2nd min) was lowest in Group 3 (1.9 μmol/min) and highest in Group 1 (8.3 μmol/min). No difference in lactate release was observed. Release of glutathione during the first 5 min of reperfusion was 43.8 ± 7.9 nmol in Group 1, but only 3.6 ± 0.7 in Group 2 (p < 0.05). Conclusions: In isolated guinea pig hearts, controlled oxygen delivery during post-ischemic reperfusion by both, reduction of coronary flow and PO₂, improves recovery of pump function. The effect is accompanied by less oxidative stress, as indicated by lowered rates of glutathione release. Received: 1 December 1998, Returned for 1. revision: 4 January 1999, 1. Revision received: 28 January 1999, Returned for 2. revision: 8 February 1999, 2. Revision received: 18 February 1999, Accepted: 2 March 1999     

Electrophysiological characterization of murine myocardial ischemia and infarction

Background Genetically altered mice will provide important insights into a wide variety of processes in cardiovascular physiology underlying myocardial infarction (MI). Comprehensive and accurate analyses of cardiac function in murine models require implementation of the most appropriate techniques and experimental protocols. Objective In this study we present in vivo, whole-animal techniques and experimental protocols for detailed electrophysiological characterization in a mouse model of myocardial ischemia and infarction. Methods FVB mice underwent open-chest surgery for ligation of the left anterior descending coronary artery or sham-operation. By means of echocardiographic imaging, electrocardiography, intracardiac electrophysiology study, and conscious telemetric ECG recording for heart rate variability (HRV) analysis, we evaluated ischemic and post-infarct cardiovascular morphology and function in mice. Results Coronary artery ligation resulted in antero-apical infarction of the left ventricular wall. MI mice showed decreased cardiac function by echocardiography, infarct-typical pattern on ECG, and increased arrhythmia vulnerability during electrophysiological study. Electrophysiological properties were determined comprehensively, but were not altered significantly as a consequence of MI. Autonomic nervous system function, measured by indices of HRV, did not appear altered in mice during ischemia or infarction. Conclusions Cardiac conduction, refractoriness, and heart rate variability appear to remain preserved in a murine model of myocardial ischemia and infarction. Myocardial infarction may increase vulnerability to inducible ventricular tachycardia and atrial fibrillation, similarly to EPS findings in humans. These data may be of value as a reference for comparison with mutant murine models necessitating ischemia or scar to elicit an identifiable phenotype. The limitations of directly extrapolating murine cardiac electrophysiology data to conditions in humans need to be considered. Received: 5 October 2000, Returned for 1. revision: 2 November 2000, 1. Revision received: 24 November 2000, Returned for 2. revision: 28 November 2000, 2. Revision received: 13 December 2000, Accepted: 14 December 2000     

Cesium chloride induced ventricular arrhythmias in dogs: three-dimensional activation patterns and their relation to the cesium dose applied

Introduction: Cesium chloride has widely been used in experimental models to produce various ventricular arrhythmias. The study was designed to evaluate whether type and mechanism of these arrhythmias are dose-dependent. Methods: In 7 dogs with acute AV-block, 60 pins containing 4 bipolar electrodes each were inserted into both ventricles to provide 240 endo-, epi- and midmyocardial recording sites. A computerized mapping system was used to determine three-dimensional activation patterns of ventricular arrhythmias induced by three injections of 1 mmol/kg cesium chloride at 20 minute intervals. Results:Out of all arrhythmias induced, 25 ventricular extrasystoles, 31 monomorphic and 47 polymorphic ventricular tachycardias were mapped. Nonsustained ventricular tachycardias were readily inducible by a single bolus of cesium chloride, whereas sustained episodes required repetitive injections (1.45 ± 0.61 vs. 2.61 ± 0.57 doses, p < 0.05). Polymorphic tachycardias were observed more commonly than monomorphic tachycardias (87 vs. 31). Initiation and maintenance of cesium induced arrhythmias were exclusively based on focal mechanisms originating from the subendocardium, irrespective of morphology and dosage. All monomorphic arrhythmias were caused by repetitive firing of single immobile foci located in either the right or the left ventricle. Bi- and multifocal mechanisms, however, were found to underlie the polymorphic episodes. Conclusions: Although there is a dose-dependence as to the sustenance of mono- or polymorphic tachycardias, this does not reflect on the three-dimensional activation pattern of cesium induced arrhythmias, which are due to mono- or multifocal activation originating from the subendocardium. Received: 6 August 1999, Returned for 1. revision: 8 October 1999, 1. Revision received: 27 October 1999, Returned for 2. Revision: 24 November 1999, 2. Revision received: 9 December 1999, Accepted: 9 December 1999     

Immunocytochemical evidence for inducible nitric oxide synthase and cyclooxygenase-2 expression with nitrotyrosine formation in human hibernating myocardium

Background: Myocardial hibernation may result from repetitive episodes of transient ischaemia leading to prolonged dysfunction. Inducible nitric oxide synthase (iNOS) expression has been demonstrated in animals following brief, non-lethal ischaemia-reperfusion injury. We therefore, hypothesised that in human hibernating myocardium: 1) iNOS would be present; 2) the reaction of nitric oxide and superoxide would form the strong oxidant peroxynitrite; 3) that this process would be accompanied by the expression of cyclooxygenase-2 (Cox-2) which interacts with NOS and whose products could further affect myocardial function. Method and results: In sixteen patients with coronary artery disease (CAD), left ventricular biopsies were obtained from chronically dysfunctional segments subtended by a stenotic artery (> 75 %) and shown to be viable by ¹⁸F-fluorodeoxyglucose positron emission tomography. Comparison was made with myocardial biopsies (n = 8) from normally contracting myocardium in patients undergoing coronary surgery, from unused transplant donors and at post-mortem. Regional wall motion score improved in all patients 6 months post-revascularisation (from 2.7 ± 0.7 to 1.5 ± 0.5; p < 0.001), confirming hibernation. Immunocytochemistry localized reactivity to iNOS, Cox-2 and nitrotyrosine (a marker of peroxynitrite formation) to cardiomyocytes from hibernating segments. No difference in reactivity to endothelial NOS was seen between hibernating and control cardiomyocytes. Conclusion: Cox-2 and iNOS are co-expressed in hibernating myocardium with nitrotyrosine suggesting nitric oxide production and peroxynitrite formation. We propose that this is secondary to ischaemia-reperfusion and that the products of these enzymes may have consequences for myocardial contractile function and survival. Received: 11 February 2002, Returned for revision: 14 February 2002, Revision received: 4 March 2002, Accepted: 11 March 2002     

Influence of perfusate calcium concentration on the inotropic insulin effect in isolated guinea pig and rat hearts

Conflicting data on the inotropic effect of insulin are present in the literature suggesting a positive inotropic property or no inotropic effect or even a negative influence. To clarify the reason for these diverging findings, dose-response curves of insulin have been performed in isolated working rat and guinea pig hearts perfused with Krebs-Henseleit buffer containing 0.9, 1.25, 2.5, and 5 mM Ca²⁺ at 37 °C. At 1.25 mM [Ca²⁺], insulin (8 to 16 IU) regularly improved the inotropic state. LVdP/dt_max increased significantly from 1,900 to 2,300 mm Hg/s (+21 %) in guinea pig hearts and from 3,197 to 4,345 mm Hg/s (+36 %) in rat hearts. LVEDP did not change significantly. Myocardial oxygen consumption increased parallel with contractility. Heart rate was not influenced in either species. Coronary flow increased by 16.5 % in guinea pig hearts, but decreased in rat hearts by 13.6 % (p < 0.05 each). With 0.9 mM [Ca²⁺] the positive inotropic effect of insulin did not further augment. At 2.5 mM [Ca²⁺] insulin exhibited in both species no significant change of LVdP/dt_max but very high insulin doses depressed the heart. At 5 mM [Ca²⁺] insulin depressed the heart significantly already at lower concentrations. At 31 °C and 1.25 mM [Ca²⁺] the positive inotropic insulin effect was preserved. We conclude that the positive inotropic insulin effect in rat and guinea pig hearts depends on the extracellular [Ca²⁺], i.e., is maximal around 1.25 mM [Ca²⁺] and is reduced or absent at higher [Ca²⁺] or may even become negative. Received: 3 September 2001/Returned for 1. revision: 24 September 2001/1. Revision received: 20 December 2001/Returned for 2. revision: 2 January 2002/2. Revision received: 21 January 2002/Accepted: 23 January 2002