Antonius Baartscheer

Contribution of Sodium Channel Mutations to Bradycardia and Sinus Node Dysfunction in LQT3 Families

Abstract— One variant of the long-QT syndrome (LQT3) is caused by mutations in the human cardiac sodium channel gene. In addition to the characteristic QT prolongation, LQT3 carriers regularly present with bradycardia and sinus pauses. Therefore, we studied the effect of the 1795insD Na+ channel mutation on sinoatrial (SA) pacemaking. The 1795insD channel was previously characterized by the presence of a persistent inward current (Ipst) at −20 mV and a negative shift in voltage dependence of inactivation. In the present study, we first additionally characterized Ipst over the complete voltage range of the SA node action potential (AP) by measuring whole-cell Na+ currents (INa) in HEK-293 cells expressing either wild-type or 1795insD channels. Ipst for 1795insD channels varied between 0.8±0.2% and 1.9±0.8% of peak INa. Activity of 1795insD channels during SA node pacemaking was confirmed by AP clamp experiments. Next, Ipst and the negative shift were implemented into SA node AP models. The −10-mV shift decreased sinus rate by decreasing diastolic depolarization rate, whereas Ipst decreased sinus rate by AP prolongation, despite a concomitant increase in diastolic depolarization rate. In combination, moderate Ipst (1% to 2%) and the shift reduced sinus rate by ≈10%. An additional increase in Ipst could result in plateau oscillations and failure to repolarize completely. Thus, Na+ channel mutations displaying an Ipst or a negative shift in inactivation may account for the bradycardia seen in LQT3 patients, whereas SA node pauses or arrest may result from failure of SA node cells to repolarize under conditions of extra net inward current.

Increased Na+/H+-exchange activity is the cause of increased [Na+]i and underlies disturbed calcium handling in the rabbit pressure and volume overload heart failure model

OBJECTIVE Cytosolic sodium ([Na+]i) is increased in heart failure (HF). We hypothesize that up-regulation of Na+/H+-exchanger (NHE) in heart failure is causal to the increase of [Na+]i and underlies disturbance of cytosolic calcium ([Ca2+]i) handling. METHODS Heart failure was induced in rabbits by combined volume and pressure overload. Age-matched animals served as control. [Na+]i, cytosolic calcium [Ca2+]i and cytosolic pH (pH(i)) were measured in isolated left ventricular midmural myocytes with SBFI, indo-1 and SNARF. SR calcium content was measured as the response of [Ca2+]i to rapid cooling (RC). Calcium after-transients were elicited by cessation of rapid stimulation (3 Hz) in the presence of 100 nmol/l noradrenalin. NHE and Na+/K+-ATPase activity were inhibited with 10 micromol/l cariporide and 100 micromol/l ouabain, respectively. RESULTS At all stimulation rates (0-3 Hz) [Na+]i and diastolic [Ca2+]i were significantly higher in HF than in control. With increasing frequency [Na+]i and diastolic [Ca2+]i progressively increased in HF and control, and the calcium transient amplitude (measured as total calcium released from SR) decreased in HF and increased in control. In HF (at 2 Hz), SR calcium content was reduced by 40% and the calcium gradient across the SR membrane by 60%. Fractional systolic SR calcium release was 90% in HF and 60% in control. In HF the rate of pH(i) recovery following acid loading was much faster at all pH(i) and NHE dependent sodium influx was almost twice as high as in control. In HF cariporide (10 micromol/l, 5 min) reduced [Na+]i and end diastolic [Ca2+]i to almost control values, and reversed the relation between calcium transient amplitude and stimulation rate from negative to positive. It increased SR calcium content and SR membrane gradient and decreased fractional systolic SR depletion to 60%. Cariporide greatly reduced the susceptibility to develop calcium after-transients. In control animals, cariporide had only minor effects on all these parameters. Increase of [Na+]i with ouabain in control myocytes induced abnormal calcium handling as found in HF. CONCLUSIONS In HF up-regulation of NHE activity is causal to increased [Na+]i and secondarily to disturbed diastolic, systolic and SR calcium handling. Specific inhibition of NHE partly normalized [Na+]i, end diastolic [Ca2+]i, and SR calcium handling and reduced the incidence of calcium after-transients. Chronic treatment with specific NHE inhibitors may provide a useful future therapeutic option in treatment of developing hypertrophy and heart failure.

Acute administration of fish oil inhibits triggered activity in isolated myocytes from rabbits and patients with heart failure

Background— Fish oil reduces sudden death in patients with prior myocardial infarction. Sudden death in heart failure may be due to triggered activity based on disturbed calcium handling. We hypothesized that superfusion with &ohgr;3-polyunsaturated fatty acids (&ohgr;3-PUFAs) from fish inhibits triggered activity in heart failure. Methods and Results— Ventricular myocytes were isolated from explanted hearts of rabbits with volume- and pressure-overload–induced heart failure and of patients with end-stage heart failure. Membrane potentials (patch-clamp technique) and intracellular calcium (indo-1 fluorescence) were recorded after 5 minutes of superfusion with Tyrode’s solution (control), &ohgr;-9 monounsaturated fatty acid oleic acid (20 &mgr;mol/L), or &ohgr;3-PUFAs (docosahexaenoic acid or eicosapentaenoic acid 20 &mgr;mol/L). &ohgr;3-PUFAs shortened the action potential at low stimulation frequencies and caused an ≈25% decrease in diastolic and systolic calcium (all P<0.05). Subsequently, noradrenalin and rapid pacing were used to evoke triggered activity, delayed afterdepolarizations, and calcium aftertransients. &ohgr;3-PUFAs abolished triggered activity and reduced the number of delayed afterdepolarizations and calcium aftertransients compared with control and oleic acid. &ohgr;3-PUFAs reduced action potential shortening and intracellular calcium elevation in response to noradrenalin. Results from human myocytes were in accordance with the findings obtained in rabbit myocytes. Conclusion— Superfusion with &ohgr;3-PUFAs from fish inhibits triggered arrhythmias in myocytes from rabbits and patients with heart failure by lowering intracellular calcium and reducing the response to noradrenalin.

[Na+](i) and the driving force of the Na+/Ca2+-exchanger in heart failure

OBJECTIVE Diastolic calcium is increased in myocytes from failing hearts despite up-regulation of the principal calcium extruding mechanism the Na+/Ca2+-exchanger (NCX). We hypothesize that increased diastolic calcium ([Ca2+]i) is secondary to increased cytosolic sodium ([Na+]i) and decreased driving force of NCX (DeltaG(exch)). METHODS The stimulation rate dependence of simultaneously measured cytosolic sodium ([Na+]i), calcium transients ([Ca2+]i) and action potentials were determined with SBFI, indo-1 and the perforated patch technique in midmural left ventricular myocytes isolated from rabbits with pressure and volume overload induced heart failure (HF) and in age matched controls. Dynamic changes of DeltaG(exch) were calculated. RESULTS With increasing stimulation frequency, 0.2-3 Hz (all data HF versus control): [Na+]i increased (6.4 to 10.8 versus 3.8 to 6.4 mmol/l), diastolic [Ca2+]i increased (142 to 219 versus 47 to 98 nmol/l), calcium transient amplitude decreased in HF (300 to 250 nmol/l) but increased in control (201 to 479 nmol/l), action potential duration (APD90) decreased (380 to 260 versus 325 to 205 ms) and time averaged DeltaG(exch) decreased (6.8 to 2.8 versus 8.7 to 6.4 kJ/mol. With increasing stimulation rate the forward mode time integral of DeltaG(exch) decreased in HF by about 30%, the reversed mode time integral increased about ninefold and the duration of reversed mode operation more than sixfold relative to control. CONCLUSIONS [Na+]i is increased in HF and the driving force of NCX is decreased. NCX exerts thermodynamic control over diastolic calcium. Disturbed diastolic calcium handling in HF is due to decreased forward mode DeltaG(exch) secondary to increased [Na+]i and prolongation of the action potential. Enhanced reversed mode DeltaG(exch) may account for increased contribution of NCX to e-c coupling in HF.

Chronic inhibition of the Na+/H+‐ exchanger causes regression of hypertrophy, heart failure, and ionic and electrophysiological remodelling

Increased activity of the Na+/H+‐exchanger (NHE‐1) in heart failure underlies raised [Na+]i causing disturbances of calcium handling. Inhibition of NHE‐1, initiated at the onset of pressure/volume overload, prevents development of hypertrophy, heart failure and remodelling. We hypothesized that chronic inhibition of NHE‐1, initiated at a later stage, would induce regression of hypertrophy, heart failure, and ionic and electrophysiological remodelling.

PGC-1α and Reactive Oxygen Species Regulate Human Embryonic Stem Cell-Derived Cardiomyocyte Function

Summary Diminished mitochondrial function is causally related to some heart diseases. Here, we developed a human disease model based on cardiomyocytes from human embryonic stem cells (hESCs), in which an important pathway of mitochondrial gene expression was inactivated. Repression of PGC-1α, which is normally induced during development of cardiomyocytes, decreased mitochondrial content and activity and decreased the capacity for coping with energetic stress. Yet, concurrently, reactive oxygen species (ROS) levels were lowered, and the amplitude of the action potential and the maximum amplitude of the calcium transient were in fact increased. Importantly, in control cardiomyocytes, lowering ROS levels emulated this beneficial effect of PGC-1α knockdown and similarly increased the calcium transient amplitude. Our results suggest that controlling ROS levels may be of key physiological importance for recapitulating mature cardiomyocyte phenotypes, and the combination of bioassays used in this study may have broad application in the analysis of cardiac physiology pertaining to disease.

Arrhythmogenesis in Heart Failure

Arrhythmogenesis in Heart Failure. In a rabbit model of heart failure produced by combined pressure and volume overload, nonsustained ventricular tachycardias developed in 15 of 23 failing rabbits. Sinus rate was increased in rabbits dying suddenly, but was decreased in survivors. This also was true in isolated preparations. Microelectrode recordings from ventricular trabeculae both from patients with end‐stage failure and from failing rabbits showed that in half of the preparations, delayed afterdepolarizations and triggered activity occurred, but only in the presence of norepinephrine and a lowered extracellular K+ concentration of 3 mM. This was due to spontaneous release of Ca2+ from the sarcoplasmic reticulum.

A Diet Rich in Unsaturated Fatty Acids Prevents Progression Toward Heart Failure in a Rabbit Model of Pressure and Volume Overload

Background— During heart failure (HF), cardiac metabolic substrate preference changes from fatty acid (FA) toward glucose oxidation. This change may cause progression toward heart failure. We hypothesize that a diet rich in FAs may prevent this process, and that dietary &ohgr;3-FAs have an added antiarrhythmic effect based on action potential (AP) shortening in animals with HF. Methods and Results— Rabbits were fed a diet containing 1.25% (w/w) high oleic sunflower oil (HF-&ohgr;9, N=11), 1.25% fish oil (HF-&ohgr;3, N=11), or no supplement (HF-control, N=8). Subsequently, HF was induced by volume and pressure overload. After 4 months, HF-parameters were assessed, electrocardiograms were recorded, and blood and ventricular tissue were collected. Myocytes were isolated for patch clamp or intracellular Ca2+- recordings to study electrophysiologic remodeling and arrhythmogenesis. Both the HF-&ohgr;9 and the HF-&ohgr;3 groups had larger myocardial FA oxidation capacity than HF control. The HF-&ohgr;3 group had significantly lower mean (± SEM) relative heart and lung weight (3.3±0.13 and 3.2±0.12 g kg−1, respectively) than HF control (4.8±0.30 and 4.5±0.23), and shorter QTc intervals (167±2.6 versus 182±6.4). The HF-&ohgr;9 also displayed a significantly reduced relative heart weight (3.6±0.26), but had similar QTc (179±4.3) compared with HF control. AP duration in the HF-&ohgr;3 group was ≈20% shorter due to increased Ito1 and IK1 and triggered activity, and Ca2+-aftertransients were less than in the HF-&ohgr;9 group. Conclusions— Dietary unsaturated FAs started prior to induction of HF prevent hypertrophy and HF. In addition, fish oil FAs prevent HF-induced electrophysiologic remodeling and arrhythmias.

Adenovirus gene transfer of SERCA in heart failure. A promising therapeutic approach

See article by Chossat et al. [10] (pages 288–297) in this issue. Abnormal and reduced contractile function is characteristic in patients suffering from heart failure. Therefore, it seems obvious that inotropic therapy could potentially be beneficial in the treatment of heart failure. However, a number of studies show short-term beneficial effects with progressive myocardial dysfunction in long-term treatment with inotropic interventions in chronic heart failure [1,2]. Inotropic interventions not only enhance contractility and cytosolic systole calcium concentration ([Ca2+]), but also potentially adversely affect diastolic [Ca2+]. Functional abnormalities commonly observed in the failing heart include a negative force frequency relationship secondary to disturbed calcium handling [3–6] characterized by increased diastolic [Ca2+], increased duration and reduced amplitude of the calcium transient and reduction of the SR calcium content [7,8]. Available evidence strongly suggests that down regulation of the sarcoplasmic reticular SR Ca-ATPase (SERCA) underlies these abnormalities possibly in combination with increased sarcolemmal Na+/Ca2+-exchange activity. Consequently, therapies aiming to enhance SERCA activity might prove potentially beneficial in the treatment of heart failure. A promising approach to this end could be adenoviral gene therapy to compensate for compromised SERCA synthesis. Indeed, one of the first studies using adenoviral gene transfer in isolated neonatal cardiac myocytes reported a successful 7.5 fold increase of total SERCA [9]. However, in similar later studies in adult normal and failing myocytes increase of SERCA was limited to maximally 1.5 to 2 fold [10–12]. Similar results were reported in transgenic animals over-expressing SERCA [12]. Over-expression of SERCA appears limited by post-transcriptional events. Although SERCA mRNA levels continuously increase with increasing viral titer, protein levels become saturated [13]. Both in healthy transgenic mice and in adenovirus treated non-failing cardiac myocytes over expression of SERCA resulted … * Tel.: +31-20-566-3265; fax: +31-20-697-5458 a.baartscheer{at}amc.uva.nl

Cellular calcium homeostasis during ischemia; a thermodynamic approach

The pivotal role of calcium cycling and homeostasis has long been recognized in contractile, metabolic, electrical and ionic alterations associated with myocardial ischemia and anoxia, as well as in hibernation, stunning and mitochondrial dysfunction associated with reperfusion. However, the lack of adequate techniques seriously hampered measurement of cellular calcium in the low and narrow range of physiological concentrations, particularly in the cytoplasm. Uninterrupted measurement of the dynamics of calcium in the cytoplasm and in organelles such as the sarcoplasmic reticulum and mitochondria proved impossible for a long time. Bourdillon and Poole-Wilson [1] were the first to accomplish this goal in 1981. They devised an elegant technique to continuously monitor mechanical function, calcium uptake and release during ischemia and reperfusion using calcium isotopes and radioactive labeling of the extracellular space. Their basic observations (see below) attracted much attention and the study remains frequently cited. Since then, new techniques have become available, superior in sensitivity and time resolution, among which are intracellular calcium specific fluorescent indicators [2–4] and 19F NMR [5,6]. Consequently, an abundance of original papers and many review articles have been published over the last 2 decades addressing the issue of calcium handling during ischemia and reperfusion. It is beyond the scope of this short update to present a full record of all literature on calcium handling during ischemia, anoxia and reperfusion over the past 2 decades. For an overview of the reperfusion related literature the reader is referred to recent review articles dealing with: the role of the Na/H-exchanger [7–9], mitochondria [10–12], sarcoplasmic reticulum [13], e–c-coupling in stunning [14], hibernation [15] and clinical implications [16]. The aim of the present contribution is to put the available data on calcium handling and homeostasis during ischemia and anoxia in a thermodynamic perspective with particular emphasis …