Bodo Brandts

Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors.

Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.

A novel membrane receptor with high affinity for lysosphingomyelin and sphingosine 1-phosphate in atrial myocytes

Activation of IK(ACh) is the major effect of the vagal neutrotransmitter acetylcholine in the heart. We report that both lysosphingomyelin (D‐erythro‐sphingosyl‐phosphorylcholine; SPC) and sphingosine 1‐phosphate (SPP) activate IK(ACh) in guinea pig atrial myocytes through the same receptor with an EC50 of 1.5 and 1.2 nM, respectively. Pertussis toxin abolished the activation of IK(ACh) by either lipid. The putative receptor showed an exquisite stereoselectivity for the naturally occurring D‐erythro‐(2S,3R)‐SPC stereoisomer, the structure of which was confirmed by mass spectroscopy and NMR. These lipids caused complete homologous and heterologous desensitization with each other but not with ACh, indicating that both act on the same receptor. This receptor displays a distinct structure‐activity relationship: it requires an unsubstituted amino group because N‐acetyl‐SPC, lysophosphatidic acid and lysophosphatidylcholine were inactive. Because SPP and SPC are naturally occurring products of membrane lipid metabolism, it appears that these compounds might be important extracellular mediators acting on a family of bona fide G protein‐coupled receptors. Expression of these receptors in the heart raises the possibility that sphingolipids may be a part of the physiological and/or pathophysiological regulation of the heart. Based on their ligand selectivity we propose a classification of the sphingolipid receptors.

Activation of muscarinic K+ current in guinea‐pig atrial myocytes by sphingosine‐1‐phosphate.

1. Activation of muscarinic K+ current (IK(ACh)) by sphingosine‐1‐phosphate (Sph‐1‐P) was studied in isolated cultured guinea‐pig atrial myocytes using whole‐cell voltage clamp. 2. Sph‐1‐P caused activation of IK(ACh) with an EC50 of 1.2 nM. The maximal current that could be activated by Sph‐1‐P amounted to about 90% of the IK(ACh) caused by a saturating concentration of acetylcholine (ACh, 10 microM). Sphingosine (1 microM), which can mimic the signalling effects of Sph‐1‐P in other cells, failed to cause measurable activation of IK(ACh). 3. IK(ACh) activation by Sph‐1‐P was completely suppressed in cells treated with pertussis toxin. 4. Desensitization of muscarinic receptors by pre‐incubation of the cells with carbachol did not affect the response to Sph‐1‐P; likewise, pre‐incubation of the cells with Sph‐1‐P resulted in a reduced sensitivity to the phospholipid but not to ACh. In contrast, pre‐incubation with either Sph‐1‐P or a serum phospholipid previously described as activating atrial IK(ACh) resulted in reduced sensitivity to both phospholipids. 5. It is concluded that activation of IK(ACh) by Sph‐1‐P in atrial myocytes is induced by binding to a novel G protein‐coupled phospholipid receptor.

Assessment of left atrial volumes in sinus rhythm and atrial fibrillation using the biplane area-length method and cardiovascular magnetic resonance imaging with TrueFISP.

OBJECTIVES To determine whether the biplane area-length method can be used for the evaluation of left atrial volumes and ejection fraction with cardiovascular magnetic resonance imaging (CMR) by TrueFISP in normal subjects and patients with atrial fibrillation. BACKGROUND Atrial fibrillation is the most common arrhythmia in elderly patients. Left atrial size and volumes play an important role in predicting short and long-term success after cardioversion. METHODS Fifteen healthy subjects (mean age 65.6+/-6.4 years) and 18 patients (mean age 67.2+/-8.8 years) with atrial fibrillation were examined by CMR (Magnetom, Siemens, Erlangen, Germany). Images were acquired by TrueFISP using the horizontal and vertical long-axis plane to measure left atrial end-diastolic and end-systolic areas and longitudinal dimensions. Volumes were determined with commercially available software. Left atrial end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and ejection fraction (EF) were determined by the biplane area-length method and compared to findings obtained by the standard short-axis method. Images were acquired and analyzed a second time in the patients with atrial fibrillation. RESULTS There was no difference in age between men and women (p=0.147) and healthy subjects and patients (p=0.128) included in the study. EDV and ESV were significantly higher and SV and EF significantly lower in patients with atrial fibrillation than in healthy subjects (p < or = 0.009), regardless of the method used. The values obtained for EDV and ESV by the biplane area-length method were significantly higher in both healthy subjects (p<0.001) and patients with atrial fibrillation (p<0.001) than those obtained by the standard short-axis approach, whereas SV (p> or = 0.057) and EF (p> or = 0.118) did not differ significantly. In the second investigation in patients with atrial fibrillation, ESV, SV, and EF did not differ significantly between the two methods (p> or =0.481). Assessment of interobserver variability revealed good agreement in the findings of the two observers, both in normal sinus rhythm and atrial fibrillation (overall variability 0.8+/-6.5%). CONCLUSIONS The biplane area-length method can be used in CMR images obtained by TrueFISP to assess left atrial volumes and ejection fraction in normal subjects and patients with varying cardiac cycle length, as in atrial fibrillation.

Arrhythmias in the intensive care patient

Purpose of reviewAtrial fibrillation, atrial flutter, AV–nodal reentry tachycardia with rapid ventricular response, atrial ectopic tachycardia, and preexcitation syndromes combined with atrial fibrillation or ventricular tachyarrhythmias are typical arrhythmias in intensive care patients. Most frequently, the diagnosis of the underlying arrhythmia is possible from the physical examination, the response to maneuvers or drugs, and the 12-lead surface electrocardiogram. In all patients with unstable hemodynamics, immediate DC-cardioversion is indicated. Conversion of atrial fibrillation to sinus rhythm is possible using antiarrhythmic drugs. Amiodarone has a conversion rate in atrial fibrillation of up to 80%. However, caution in the use of short-term administration of intravenous amiodarone in critically ill patients with recent-onset atrial fibrillation is absolutely necessary, and the duration of therapy should not exceed 24 to 48 hours. Ibutilide represents a relatively new class III antiarrhythmic agent that has been reported to have conversion rates of 50% to 70%; it seems that ibutilide is even successful when intravenous amiodarone failed to convert atrial fibrillation. Recent findingsNewer studies compared the outcome of patients with atrial fibrillation and rhythm- or rate-control. Data from these studies (AFFIRM, RACE) clearly showed that rhythm control is not superior to rate control for the prevention of death and morbidity from cardiovascular causes. Therefore, rate-control may be an appropriate therapy in patients with recurrent atrial fibrillation after DC-cardioversion. Acute therapy of atrial flutter in intensive care patients depends on the clinical presentation. Atrial flutter can most often be successfully cardioverted to sinus rhythm with energies less than 50 joules. Ibutilide trials showed efficacy rates of 38–76% for conversion of atrial flutter to sinus rhythm compared with conversion rates of 5–13% when intravenous flecainide, propafenone, or verapamil was administered. In addition, a high dose (2 mg) of ibutilide was more effective than sotalol (1.5 mg/kg) in conversion of atrial flutter to sinus rhythm (70% versus 19%). SummaryThere is general agreement that bystander first aid, defibrillation, and advanced life support is essential for neurologic outcome in patients after cardiac arrest due to ventricular tachyarrhythmias. The best survival rate from cardiac arrest can be achieved only when (1) recognition of early warning signs, (2) activation of the emergency medical services system, (3) basic cardiopulmonary resuscitation, (4) defibrillation, (5) management of the airway and ventilation, and (6) intravenous administration of medications occurs as rapidly as possible. Public access defibrillation, which places automatic external defibrillators in the hands of trained laypersons, seems to be an ideal approach in the treatment of ventricular fibrillation. The use of automatic external defibrillators by basic life support ambulance providers or first responder in early defibrillation programs has been associated with a significant increase in survival rates. Drugs such as lidocaine, procainamide, sotalol, amiodarone, or magnesium were recommended for treatment of ventricular tachyarrhythmias in intensive care patients. Amiodarone is a highly efficacious antiarrhythmic agent for many cardiac arrhythmias, ranging from atrial fibrillation to malignant ventricular tachyarrhythmias, and seems to be superior to other antiarrhythmic agents.

Downregulation of muscarinic M2 receptors linked to K+ current in cultured guinea-pig atrial myocytes.

1. Desensitization of muscarinic K+ current (IK(ACh)) was studied in cultured atrial myocytes from guinea‐pig hearts using whole‐cell voltage clamp. 2. Three different types of desensitization could be identified. A fast component which upon rapid superfusion with ACh resulted in a partial relaxation of IK(ACh) within a few seconds to a plateau which was maintained in the presence of ACh. Recovery from this type of desensitization paralleled the decay of IK(ACh) after washout of the agonist. A second type of desensitization was observed within minutes. This was reversed around 10 min after washout of ACh. Both types were heterologous with regard to the A1 receptor and the novel phospholipid (Pl) receptor, both of which activate IK(ACh) via the same signalling pathway. 3. A third type of desensitization (downregulation) occurred upon exposure of the cultures for 24‐48 h to the muscarinic agonist carbachol (CCh). The level of downregulation depended on the concentration of CCh (0.1 microM < or = [CCh] < or = 10 microM). No recovery was observed within 5 h after washout of CCh. Thereafter sensitivity to ACh slowly returned (half‐time (t1/2), approximately 20 h). 4. Downregulation by CCh (0.1‐5 microM) was characterized by an increase in EC50 for ACh with no reduction in maximum IK(ACh). With 5 microM CCh, EC50 was increased from 0.1 to 3.7 microM. At 10 microM CCh EC50 was increased to 15 microM and maximal current that could be evoked by ACh was reduced to 15%. 5. Downregulation by CCh was homologous with regard to A1 and Pl receptors. Maximum IK(ACh), assayed by a saturating concentration of Pl, was not reduced in downregulated cells, suggesting a mechanism localized at the M2 receptor. 6. The changes in the concentration‐response curves can be accounted for by assuming an excess of M2 receptors relative to the subsequent component of the signalling pathway. 7. As the intact heart is under tonic vagal control, downregulation is likely to contribute to controlling the sensitivity of the heart to vagal activity in situ.

Single and biplane TrueFISP cardiovascular magnetic resonance for rapid evaluation of left ventricular volumes and ejection fraction.

INTRODUCTION Cardiovascular magnetic resonance (CMR) allows very accurate, but time-consuming, volume assessment by the short-axis slice summation technique. The single and biplane methods of volume assessment are used less, partly because FLASH cine imaging provides poor blood-myocardium contrast in long-axis views. TrueFISP gives excellent blood-myocardium contrast, even in patients with heart failure. We hypothesized that the single plane and biplane methods of volume assessment in TrueFISP images might provide an acceptable degree of accuracy and be quicker than the short axis method, and that single and biplane left ventricular volume assessment would be more accurate with TrueFISP than with FLASH in patients with impaired ventricular function. METHODS Short- and long-axis CMR images were obtained by FLASH and TrueFISP with a 1.5-T scanner. We determined the accuracy of both single and biplane long-axis methods for left ventricular volume and ejection fraction (EF) measurements compared with the conventional short-axis method in 10 heart failure patients using both FLASH and TrueFISP and in 9 healthy subjects using TrueFISP. RESULTS No difference in volumes and EF was found between the single plane method, the biplane method, and the short-axis method using TrueFISP for image acquisition, in both patients and healthy subjects. The same was true of the results obtained by FLASH in the patients with heart failure. CONCLUSIONS The single and biplane methods, regardless of whether TrueFISP or FLASH is used, are a reasonable and rapid alternative to the conventional short-axis approach for left ventricular volume and EF assessment in patients with heart failure and impaired ventricular function.

In vivo downregulation of M2 receptors revealed by measurement of muscarinic K+ current in cultured guinea-pig atrial myocytes.

1 Muscarinic K+ current (IK(ACh)) elicited by acetylcholine (ACh) was measured in guinea‐pig atrial myocytes, which were either freshly isolated or cultured for up to 8 days. 2 The half‐time of activation of inward IK(ACh) by a saturating concentration (10 μm) of ACh decreased from ∼400 ms (in freshly isolated cells) to 250 ms after 6 days in culture. This was paralleled by an increase in the fast desensitizing component of IK(ACh). The density of steady‐state currents was not changed. Downregulation of M2 receptors by long‐term treatment of isolated myocytes with carbachol in vitro had opposite effects. 3 The EC50 of ACh for the activation of steady‐state −K(ACh) was reduced from 5 × 107 M (day 0) to 8 × 108 m (day 6). The shift in EC50 occurred with a half‐time of about 2 days, similar to the recovery from downregulation induced by treating atrial myocytes with carbachol in vitro. 4 The increase in sensitivity to ACh can be accounted for by an ∼6‐fold increase in the density of M2 receptors. 5 It is concluded that sensitization in culture reflects recovery from downregulation of M2 receptors due to the tonic vagal input to the heart in the intact animal.

Antisense oligonucleotides against receptor kinase GRK2 disrupt target selectivity of β‐adrenergic receptors in atrial myocytes

K+ channels composed of GIRK subunits are predominantly expressed in the heart and various regions of the brain. They are activated by βγ‐subunits released from pertussis toxin‐sensitive G‐proteins coupled to different seven‐helix receptors. In rat atrial myocytes, activation of K(ACh) channels is strictly limited to receptors coupled to pertussis toxin‐sensitive G‐proteins. Upon treatment of myocytes with antisense oligodesoxynucleotides against GRK2, a receptor kinase with Gβγ binding sites, in a fraction of cells, K(ACh) channels can be activated by β‐adrenergic receptors. Sensitivity to β‐agonist is insensitive to pertussis toxin treatment. These findings demonstrate a potential role of Gβγ binding proteins for target selectivity of G‐protein‐coupled receptors

Non-receptor-mediated activation of IK(ATP) and inhibition of IK(ACh) by diadenosine polyphosphates in guinea-pig atrial myocytes

1 The effects of diadenosine polyphosphates (APnA, where n= 4–6) were studied on beating frequency of perfused guinea‐pig hearts and on muscarinic K+ current (IK(ACh)) and ATP‐regulated K+ current (IK(ATP)) in atrial myocytes from guinea‐pig hearts using whole‐cell voltage clamp. 2 Bradycardia induced by APnA in perfused hearts was completely inhibited by 8‐cyclopentyl‐ 1,3‐dipropylxanthine (CPX, 20 μm), a selective antagonist at A1 adenosine receptors, and was augmented by dipyridamole (Dipy), an inhibitor of cellular adenosine (Ado) uptake. 3 Whereas exposure of atrial myocytes to Ado (100 μm) within about 1 s induced a significant whole‐cell IK(ACh), APnA up to 1 mm applied for some tens of seconds failed to activate IK(ACh). If present for periods > 2 min, APnA caused inhibition of agonist‐evoked IK(ACh) and activation of a weakly inward rectifying K+ current, which was identified as IK(ATP) by its sensitivity to glibenclamide and its current‐voltage curve. 4 The actions of extracellular APnA on IK(ACh) and IK(ATP) were mimicked by intracellular loading of compounds via the patch clamp pipette and by intracellular loading of AMP. 5 The results from isolated myocytes exclude APnA acting as A1 agonists. It is suggested that myocytes can take up APnA, which are degraded to AMP. In the presence of ATP, AMP is converted to ADP, a physiological activator of ATP‐regulated K+ channels, by adenylate kinase. A similar mechanism resulting in a reduction of the [GTP]/[GDP] ratio might be responsible for inhibition of IK(ACh). 6 In the perfused heart and other multicellular cardiac preparations the actions of APnA are mediated by Ado via A1 receptors. It is suggested that APnA in multicellular cardiac tissue are hydrolysed by an ectohydrolase to yield AMP which is converted to Ado by ectonucleotidases.