Ventricular repolarization time, location of pacing stimulus and current pulse amplitude conspire to determine arrhythmogenicity in mice.

AIM: In this study, we investigate the impact of altered action potential durations (APD) on ventricular repolarization time and proarrhythmia in mice with and without genetic deletion of the K+ -channel-interacting protein 2 (KChIP2-/- and WT respectively). Moreover, we examine the interrelationship between the dispersion of repolarization time and current pulse amplitude in provoking ventricular arrhythmia. METHODS: Intracardiac pacing in anesthetized mice determined refractory periods and proarrhythmia susceptibility. Regional activation time (AT), APD and repolarization time (=AT + APD) were measured in isolated hearts using floating microelectrodes. RESULTS: Proarrhythmia in WT and KChIP2-/- was not sensitive to changes in refractory periods. Action potentials were longer in KChIP2-/- hearts compared to WT hearts. Isolated WT hearts had large apico-basal dispersion of repolarization time, whereas hearts from KChIP2-/- mice had large left-to-right ventricular dispersion of repolarization time. Pacing from the right ventricle in KChIP2-/- mice in vivo revealed significant lower current pulse amplitudes needed to induce arrhythmias in these mice. CONCLUSION: Large heterogeneity of repolarization time is proarrhythmic when pacing is delivered from the location of earlier repolarization time. Ventricular repolarization time, location of the pacing stimulus and the amplitude of the stimulating current pulse are critical parameters underlying arrhythmia vulnerability.

A kinase inhibitor screen identifies Mcl-1 and Aurora kinase A as novel treatment targets in antiestrogen-resistant breast cancer cells.

Antiestrogen resistance is a major problem in breast cancer treatment. Therefore, the search for new therapeutic targets and biomarkers for antiestrogen resistance is crucial. In this study, we performed a kinase inhibitor screen on antiestrogen responsive MCF-7 cells and a panel of MCF-7-derived tamoxifen- and fulvestrant-resistant cell lines. Our focus was to identify common and distinct molecular mechanisms involved in tamoxifen- and fulvestrant-resistant cell growth. We identified 18 inhibitors, of which the majority was common for both tamoxifen- and fulvestrant-resistant cell lines. Two compounds, WP1130 and JNJ-7706621, exhibiting prominent preferential growth inhibition of antiestrogen-resistant cell lines, were selected for further studies. WP1130, a deubiquitinase inhibitor, induced caspase-mediated cell death in both tamoxifen- and fulvestrant-resistant cell lines by destabilization of the anti-apoptotic protein Mcl-1. Mcl-1 expression was found upregulated in the antiestrogen-resistant cell lines and depletion of Mcl-1 in resistant cells caused decreased viability. JNJ-7706621, a dual Aurora kinase and cyclin-dependent kinase inhibitor, specifically inhibited growth and caused G2 phase cell cycle arrest of the tamoxifen-resistant cell lines. Knockdown studies showed that Aurora kinase A is essential for growth of the tamoxifen-resistant cells and inhibition of Aurora kinase A resensitized tamoxifen-resistant cells to tamoxifen treatment. Preferential growth inhibition by WP1130 and JNJ-7706621 was also found in T47D-derived tamoxifen-resistant cell lines, pointing at Mcl-1 and Aurora kinase A as potential treatment targets. In addition, tumor samples from 244 estrogen receptor-positive breast cancer patients treated with adjuvant tamoxifen showed that higher expression level of Aurora kinase A was significantly associated with shorter disease-free and overall survival, demonstrating the potential of Aurora kinase A as a biomarker for tamoxifen resistance.

Physiology and analysis of the electrocardiographic T wave in mice.

AIM: The murine electrocardiogram (ECG) is a valuable tool in cardiac research, although the definition of the T wave has been a matter of debate for several years potentially leading to incomparable data. By this study, we seek to make a clear definition of the murine T wave. Moreover, we investigate the consequences of performing QT interval correction in anaesthetized mice. METHODS: Electrocardiograms from conscious mice were recorded by implanted telemetry devices. Surface ECGs were recorded from anaesthetized mice before and during pharmacological interventions, ventricular ischaemia and heart failure. Right atrial pacing was performed to evaluate the relationship between heart rate and QT intervals. RESULTS: Electrocardiogram traces of conscious and anaesthetized mice (lead II) showed separable positive J waves and negative T waves. The end of the T wave was determined as the point where the T wave returned to the isoelectric line. Atrial pacing revealed that the duration of the QT interval is independent of heart rate in anaesthetized mice. The calcium channel blocker, verapamil, prolonged the PR interval; however, the polarities of the J and T waves were not changed. Local cardiac ischaemia and ß-adrenergic stimulation caused indistinguishable positive J and T waves. In contrast, chronic heart failure caused entirely negative J and T waves. In every case, the end of the T wave was clearly distinguishable on the ECG. CONCLUSION: The end of the T wave is readily available from conscious and anaesthetized mice. Heart rate correction of QT interval duration in the anaesthetized mouse is not recommended.

Comparison of the IKr blockers moxifloxacin, dofetilide and E-4031 in five screening models of pro-arrhythmia reveals lack of specificity of isolated cardiomyocytes.

BACKGROUND AND PURPOSE: Drug development requires the testing of new chemical entities for adverse effects. For cardiac safety screening, improved assays are urgently needed. Isolated adult cardiomyocytes (CM) and human embryonic stem cell-derived cardiomyocytes (hESC-CM) could be used to identify pro-arrhythmic compounds. In the present study, five assays were employed to investigate their sensitivity and specificity for evaluating the pro-arrhythmic properties of I(Kr) blockers, using moxifloxacin (safe compound) and dofetilide or E-4031 (unsafe compounds). EXPERIMENTAL APPROACH: Assays included the anaesthetized remodelled chronic complete AV block (CAVB) dog, the anaesthetized methoxamine-sensitized unremodelled rabbit, multi-cellular hESC-CM clusters, isolated CM obtained from CAVB dogs and isolated CM obtained from the normal rabbit. Arrhythmic outcome was defined as Torsade de Pointes (TdP) in the animal models and early afterdepolarizations (EADs) in the cell models. KEY RESULTS: At clinically relevant concentrations (5-12 µM), moxifloxacin was free of pro-arrhythmic properties in all assays with the exception of the isolated CM, in which 10 µM induced EADs in 35% of the CAVB CM and in 23% of the rabbit CM. At supra-therapeutic concentrations (≥100 µM), moxifloxacin was pro-arrhythmic in the isolated rabbit CM (33%), in the hESC-CM clusters (18%), and in the methoxamine rabbit (17%). Dofetilide and E-4031 induced EADs or TdP in all assays (50-83%), and the induction correlated with a significant increase in beat-to-beat variability of repolarization. CONCLUSION AND IMPLICATIONS: Isolated cardiomyocytes lack specificity to discriminate between TdP liability of the I(Kr) blocking drugs moxifloxacin and dofetilide or E4031.

Double pharmacological challenge on repolarization opens new avenues for drug safety research.

The pharmaceutical industry is testing new potential drugs for their propensity to prolong human cardiac repolarization, and regards this as a sign of proarrhythmic risk. Many studies have dethroned the common perception that prolonged repolarization is a reliable surrogate marker for torsades de pointes (TdP) arrhythmia. Both the pharmaceutical industry and the regulatory bodies are neglecting the available proarrhythmia models. In vitro studies have suggested that combined pharmacological hits on repolarization will produce a superior substrate for in vivo proarrhythmia, compared to the single-drug assessment. By using consecutive pharmacological challenges, a simple model is proposed, in which combinatorial pharmacology is employed to provoke TdP in the conscious dog. The pharmaceutical industry interested in evaluating the proarrhythmic potential of their present and future drugs now has a simple means of doing so.

No proarrhythmic properties of the antibiotics Moxifloxacin or Azithromycin in anaesthetized dogs with chronic-AV block.

BACKGROUND & PURPOSE: The therapeutically available quinolone antibiotic moxifloxacin has been used as a positive control for prolonging the QT interval in both clinical and non-clinical studies designed to assess the potential of new drugs to delay cardiac repolarization. Despite moxifloxacin prolonging QT, it has not been shown to cause torsades de pointes arrhythmias (TdP). Azithromycin is a macrolide antibiotic that has rarely been associated, clinically, with cases of proarrhythmia. As there is a lack of clinical data available, the cardiac safety of these drugs was assessed in a TdP-susceptible animal model by evaluating their repolarization and proarrhythmia effects. EXPERIMENTAL APPROACH & KEY RESULTS: In transfected HEK cells, the IC(50)s for I (hERG) were 45+/-6 and 856+/-259 microg ml(-1) for moxifloxacin and azithromycin, respectively. Intravenous administration of 2 and 8 mg kg(-1) moxifloxacin (total peak-plasma concentrations 4.6+/-1.5 and 22.9+/-6.8 microg ml(-1)) prolonged the QT(c) in 6 anaesthetized dogs with chronic AV block by 7+/-3 and 21+/-19%, respectively. Similar intravenous doses of azithromycin (total peak-plasma concentrations 5.4+/-1.3 and 20.8+/-4.9 microg ml(-1)) had no electrophysiological effects in the same dogs. The reference compound, dofetilide (25 microg kg(-1) i.v.) caused QT(c) prolongation (29+/-15%) and TdP in all dogs. Beat-to-beat variability of repolarization (BVR), quantified as short-term variability of the left ventricular monophasic action potential duration, was only increased after dofetilide (1.8+/-0.7 to 3.8+/-1.5 ms; P<0.05). CONCLUSION & IMPLICATIONS: As neither moxifloxacin nor azithromycin caused TdP or an increase in the BVR, we conclude that both drugs can be used safely in clinical situations.

A 1-year follow-up quality of life study after hemodynamically successful or unsuccessful surgical revascularization of lower limb ischemia.

PURPOSE: The impact of hemodynamically successful or unsuccessful bypass grafting or angioplasty on patients' quality of life was assessed throughout the first year postsurgery. METHODS: A total of 146 patients, 97 patients who underwent successful revascularization and 49 patients who underwent unsuccessful revascularization, were assessed for quality of life with the Nottingham Health Profile. RESULTS: Hemodynamically successful revascularization resulted in an immediate and lasting impact on the patients' quality of life. Despite hemodynamic failure, patients had improvements in pain, emotional reactions, sleep, and family relationships at the 1-year follow-up assessment. A successful revascularization in patients with claudication demonstrated the most marked quality of life benefits, including all health dimensions that were not normal at baseline. Patients with critical ischemia had improved quality of life for pain, sleep, and physical mobility. High ankle pressure, in the claudicant group, and a high sense of coherence were significantly associated with high quality of life. CONCLUSION: The treatment of lower limb ischemia resulted in an immediate and relatively lasting improvement in patients' quality of life. Patients who underwent hemodynamically successful bypass grafting procedures or angioplasty demonstrated higher quality of life benefits than patients who underwent a failed bypass grafting procedure. Quality of life was further determined by means of the patients' sense of coherence.

The effects of successful intervention on quality of life in patients with varying degrees of lower-limb ischaemia.

OBJECTIVES: to assess the quality of life after successful intervention among patients with varying degrees of lower-limb ischaemia in comparison with healthy controls and the respondents>> degree of sense of coherence. MATERIALS AND METHODS: one hundred and twelve patients and 102 healthy controls were assessed for quality of life (Nottingham Health Profile) and sense of coherence. MAIN RESULTS: successful angioplasty or surgical intervention led to an improved quality of life at 6 months, in particular with regard to pain, sleep, physical mobility, hobbies and holiday and to a level similar to healthy controls in sleep, social isolation, paid employment and family relationships. It remained at a significantly lower level than that of healthy controls with regard to pain, emotional reactions, physical mobility, energy, housework, hobbies, holidays, sex and social life. Critical ischaemia patients did not reach the same level of quality of life as the claudicants or the healthy controls. CONCLUSION: successful treatment for chronic limb ischaemia improved the quality of life significantly, more so in claudicants than in patients with critical ischaemia. The degree to which the quality of life improved was associated with the patients>> sense of coherence and their ankle pressure.

Quality of life associated with varying degrees of chronic lower limb ischaemia: comparison with a healthy sample.

OBJECTIVES: To assess quality of life in patients with varying degrees of ischaemia in comparison with controls, and to determine whether the degree of lower limb ischaemia and sense of coherence were associated with quality of life. MATERIALS AND METHODS: 168 patients, including 93 claudicants and 75 patients with critical ischaemia and 102 controls were studied. Quality of life was assessed using the Nottingham Health Profile in addition to the Sense of Coherence scale. MAIN RESULTS: Patients with lower limb ischaemia scored significantly reduced quality of life in all aspects compared to controls. Pain, physical mobility and emotional reactions were the significant independent factors when using logistic regression analysis. The grade of disease and low sense of coherence were significantly associated with low quality of life. Increasing lower limb ischaemia significantly conferred worse pain, sleeping disturbances and immobility. CONCLUSION: This study showed that the quality of life was impaired among patients with lower limb ischaemia, in all investigated respects. The degree to which quality of life was affected seems to represent an interplay between the grade of ischaemia and the patient's sense of coherence. This suggests the need for a multidimensional assessment prior to intervention.

Local intra-arterial thrombolysis with urokinase combined with balloon angioplasty in the lower extremities.

OBJECTIVE: To assess the effect of thrombolysis with urokinase in the treatment of acute and subacute arterial thrombosis or graft occlusion. DESIGN: Open study. SETTING: County hospital, Sweden. SUBJECTS: 20 selected patients with lower limb arterial or graft occlusions of less than six months' duration, 17 of whom presented with rest pain (four with ulceration) and the rest with claudication. INTERVENTIONS: High dose urokinase (4,000 IU/minute for up to 8 hours) given intra-arterially, followed by oral anticoagulation for 6 months. MAIN OUTCOME MEASURES: Patency at one month and one year, morbidity and mortality. RESULTS: At one month 6/17 who presented with rest pain could walk unlimited distances, 8 had claudication between 50 and 500 m, and 3 had no improvement; 2 had had below knee amputations. At one year only 4 could walk unlimited distances, 5 had claudication between 50 and 500 m, 2 had rest pain, 4 had had major amputations, and 1 was dead and 1 was lost to follow up. Five patients had had 10 additional procedures. Of the 3 who presented with claudication, 2 improved their walking distance to at least 100 m, and one had total relief of symptoms after one month; after a year one had no symptoms, one had mild claudication, and one had severe claudication (120 m). Three developed complications: one bleeding 12 hours after treatment was successfully treated by transfusion, one embolism to the midpopliteal artery was successfully treated by embolectomy, and one episode of bleeding during lysis ceased when treatment was stopped. CONCLUSION: Thrombolysis is at best only an adjunct to balloon angioplasty or traditional vascular operations.

Fatal pulmonary embolism in an unselected series: the possible role of caval filters in prevention.

OBJECTIVE: To evaluate the possible benefit of inserting a caval filter for the prevention of pulmonary embolism (PE) in an unselected group of patients admitted to hospital. DESIGN: Analysis of clinical data collected prospectively. SETTING: University hospital, Sweden. SUBJECTS: 1391 patients who presented during 1987 435 with deep vein thrombosis (DVT), thrombus in the right atrium or the right ventricle or PE at necropsy; 366 patients shown to have DVT on phlebography; 44 shown to have PE on pulmonary scintigraphy; and 546 patients operated on for hip fractures. OUTCOME MEASURES: Clinical description of patients and groups of patients who died of PE who could possibly have benefitted from insertion of a filter. RESULTS: Of the 435 patients with DVT or PE or both, 141 had DVT in the femoral or iliac veins or in the inferior vena cava together with PE which contributed to the cause of death. Only 11 of these had been suspected clinically of having DVT in or below the inferior vena cava and only 1 of these was less than 80 years old and did not have malignant disease with distant metastases. Of the 366 patients with phlebographically confirmed DVT 8 died with PE that had contributed to the cause of death; 6 had incurable cancer and the remaining two were over 85 years. Of the 44 patients with scintigraphically confirmed PE 2 patients (aged 78 and 89) died of PE that had already been diagnosed clinically and none would have benefited from insertion of a filter after diagnosis. There were 9 PE deaths related to PE among 546 patients operated on for hip fractures, and all but one had potentially lethal coexisting disease or were over 80 years old. CONCLUSION: In this epidemiological survey only a few patients would have benefited from insertion of a caval filter. The results call for a restricted use of caval filters until benefit has been confirmed by prospective studies.

Changes in skin perfusion after sympathetic block with guanethidine. Laser Doppler flowmetry in human volunteers.

Total forearm blood flow and skin microcirculation have been measured by occlusion plethysmography and by laser Doppler flowmetry (skin blood cell flux) before and after the induction of regional sympathetic block with Guanethidine. Total forearm blood flow more than doubled while the skin blood cell flux increased by 50% comparing simultaneous flows of blocked and control arms. The increase lasted for 3 days during which period the flux pattern registered by the laser Doppler flowmeter in the blocked extremities showed regular variations oscillating around the mean flux in an almost sinus wave fashion. In non-blocked arms the flux pattern registered by the laser Doppler flowmeter was highly irregular. We conclude that regional sympathetic block with Guanethidine results in an increased skin microcirculation in healthy human volunteers and that the increase lasts for 3 days. No valid conclusions can be made from this study concerning the nutritive benefit of the microcirculatory change.

Bilateral ankle skin temperature as a predictor of early vascular graft patency.

The efficacy of postoperative bilateral ankle skin temperature as a predictor of early graft patency was prospectively evaluated in 138 consecutive cases of femoropopliteal bypass surgery. The difference between ipsilateral and contralateral ankle temperature (hourly registrations) was significantly greater in the patients with patent graft on postoperative day 1 than in those with an occluded graft. In measurements 8 h postoperatively, highest sensitivity (84 per cent) and specificity (93 per cent) of the test were obtained when the difference was +0.4 degrees C. The overall accuracy of prediction with this simple method was 89 per cent.

Patency, blood flow and histologic characteristics in end-to-end and end-to-side arteriovenous fistulas. Comparisons in an experimental model.

Experimental arteriovenous fistulas of end-to-end (ETE) and end-to-side (ETS) type were created with microsurgical technique in rabbits between the carotid artery and the facial vein. Blood pressure and blood flow through the fistulas were registered during the operation and the flow capacity (flow/BP) was calculated. Evaluation was made at 2 hours, 1 day, 1 month and 3 months. Immediately after completion of the anastomosis there was no statistically significant difference in blood flow capacity between the ETE and the ETS group. At 1 month the flow capacity had increased significantly in the ETS, but not in the ETE group. At 3 months 3/5 of the ETS anastomoses remained patent, but all the ETE types were occluded. Histologic findings included severe intimal thickening at the anastomotic level and on the venous side of the anastomosis at 1 and 3 months. The study indicated that late blood flow and patency may be greater in ETS than in ETE arteriovenous fistulas.

Evaluation of clinical examination preceding surgical treatment of AV-fistula problems. Is angiography necessary?

Clinical and angiographic findings at preoperative assessment were compared in a prospective investigation of 34 consecutive uraemic patients with problems of vascular access. The series comprised 26 patients with malfunctioning arteriovenous fistulas (AVF) and 8 who were about to receive a new AVF. The diagnostic sensitivity and specificity of clinical examination in malfunctioning AVF were, respectively, 50 and 100% in cases with significant stenosis upstream from the cannulation site (inflow restriction). The corresponding figures in cases with significant stenosis or vein occlusion downstream from the cannulation site (outflow restriction) were 38 and 43%. Clinical disclosure of outflow restriction is considerably impeded when there is concurrent inflow restriction. The advantages and disadvantages of the most commonly used angiographic techniques are discussed.

What causes the failures in surgically constructed arteriovenous fistulas?

A retrospective analysis was made of 191 uraemic patients whose first arteriovenous fistula was constructed in the period 1972-1978. Only direct-type lower forearm fistulas were considered. The aim was to identify and evaluate factors influencing fistula patency rate. Evaluation was done by simple and multiple regression analyses and by actuarial life table computation. Early failure of fistula occurred most frequently in patients with diabetes, peroperative blood pressure less than 110 mmHg, small-calibre veins and/or less than maximal uraemia. The early failure rate among patients with low peroperative blood pressure was 53%, and in the other patients 24%. Among patients with serum creatinine higher than 1 140 mumol/l the corresponding figure was 11%, while in those with lower values it was 35%. Long-term fistula patency was influenced by sex (with advantage to males), by diabetes or diabetic vasculopathy, and seemingly be a tendency to hypotension in some patients. Direct-type forearm fistula may well be utilized in patients with narrow veins. The operation should not be performed long before induction of haemodialysis. This is because some of the fistula's lifespan will be wasted if it is created too soon, and because the early failure rate is lower in patients with advanced uraemia. The analysis also reflects problems that can be anticipated, as diabetics constitute an increasing proportion of patients requiring vascular access.

A grafted arteriovenous fistula between the subclavian artery and ipsilateral internal jugular vein. Case report.

The case history of a haemodialysis patient with profound hypotension and persistent failures of all blood access devices is presented. A central, high flow, grafted arteriovenous fistula (PTFE) between the subclavian artery and the ipsilateral internal jugular vein is described, and its advantages over previously described central high flow arteriovenous fistulas are discussed.