Fons Verdonck

Enhanced Ca(2+) release and Na/Ca exchange activity in hypertrophied canine ventricular myocytes: potential link between contractile adaptation and arrhythmogenesis.

BackgroundVentricular arrhythmias are a major cause of sudden death in patients with heart failure and hypertrophy. The dog with chronic complete atrioventricular block (CAVB) has biventricular hypertrophy and ventricular arrhythmias and is a useful model to study underlying cellular mechanisms. We investigated whether changes in Ca2+ homeostasis are part of the contractile adaptation to CAVB and might contribute to arrhythmogenesis. Methods and ResultsIn enzymatically isolated myocytes, cell shortening, Ca2+ release from the sarcoplasmic reticulum (SR), and SR Ca2+ content were enhanced at low stimulation frequencies. Ca2+ influx through L-type Ca2+ channels was unchanged, but Ca2+ influx via the Na/Ca exchanger was increased and contributed to Ca2+ loading of the SR. Inward Na/Ca exchange currents were also larger. Changes in Ca2+ fluxes were less pronounced in the right versus left ventricle. ConclusionsEnhanced Na/Ca exchange activity may improve contractile adaptation to CAVB but at the same time facilitate arrhythmias by (1) increasing the propensity to Ca2+ overload, (2) providing more inward current leading to (nonhomogeneous) action potential prolongation, and (3) enhancing (arrhythmogenic) currents during spontaneous Ca2+ release.

Altered Na/Ca exchange activity in cardiac hypertrophy and heart failure: a new target for therapy?

Increased Na/Ca exchange (NCX) expression may be part of the genetic reprogramming in cardiac remodeling. In this review we address the following questions: (1) Is increased NCX activity a general feature of cardiac remodeling in hypertrophy and heart failure? (2) How does this contribute to the contractile and electrical phenotype of hypertrophy and heart failure? (3) Should be consider NCX a potential therapeutic target? From a review of the literature we found that NCX activity can be increased, unchanged, or even downregulated during cardiac remodeling. When NCX activity is increased, it can be considered compensatory for contractile function, but with negative side-effects, including an increased risk of arrhythmias. Changes in activity do not necessarily reflect changes in gene expression. Altered NCX activity can also be a consequence of changes in other Ca(2+) fluxes or in [Na(+)](i) homeostasis. The role of NCX in contractile alterations and arrhythmogenesis varies with the different stimuli or stages of cardiac remodeling. Pharmacological block of NCX in heart failure or hypertrophy may thus be useful, but most likely only in specific conditions, perhaps as part of a combined approach. Development of drugs that target only a specific mode of the exchanger may offer a further advantage.

Evolutionary origin of inhibitor cystine knot peptides

The inhibitor cystine knot (ICK) fold is an evolutionarily conserved structural motif shared by a large group of polypeptides with diverse sequences and bioactivities. Although found in different phyla (animal, plant, and fungus), ICK peptides appear to be most prominent in venoms of cone snail and spider. Recently, two scorpion toxins activating a calcium release channel have been found to adopt an ICK fold. We have isolated and identified both cDNA and genomic clones for this family of ICK peptides from the scorpion Opistophthalmus carinatus. The gene characterized by three well‐delineated exons respectively coding for three structural and functional domains in the toxin precursors illustrates the correlation between exon and module as suggested by the “exon theory of genes.” Based on the analysis of precursor organization and gene structure combined with the 3‐D fold and functional data, our results highlight a common evolutionary origin for ICK peptides from animals. In contrast, ICK peptides from plant and fungus might be independently evolved from another ancestor.

Intracellular Na in animal models of hypertrophy and heart failure: contractile function and arrhythmogenesis

Time for primary review 26 days. Myocardial hypertrophy (Hyp) and heart failure (HF) are pathologic states characterized by altered intracellular Ca handling [1,2] that can contribute to diastolic and/or systolic dysfunction and arrhythmias [1,3]. However, there is an important interplay between intracellular Na ([Na]i) and Ca handling, so that altered levels of [Na]i and Na transporters can have profound effects on both contractile function and arrhythmogenesis. Both intracellular \[Ca\] ([Ca]i) and intracellular pH (pHi) in cardiac myocytes depend strongly on [Na]i [1]. This is because Na/Ca exchange (NCX) and Na/H exchange (NHE) are powerful transport mechanisms that use the energy stored in the transmembrane [Na] electrochemical gradient to extrude Ca and protons from the cell. Thus, when [Na]i rises it can limit the ability of NCX and NHE to extrude Ca and protons from myocytes. This could slow relaxation and recovery of pHi from acid loads (e.g. during ischemia). First let us consider whether [Na]i is altered in hypertrophy and HF. Numerous reports indicate that [Na]i is increased in hypertrophy [4–7]. The magnitude of [Na]i elevation (4–6 mM) in hypertrophied guinea pig hearts (induced by aortic banding) was consistent, whether measured by ion-sensitive electrodes (ISE) [4] or nuclear magnetic resonance [5]. Additionally, myocytes from hypertrophied rat hearts (induced by isoproterenol infusion) exhibit a ∼6 mM increase in [Na]i assessed by ISE [6]. Dogs with hypertrophy induced by chronic AV block (cAVB) demonstrate a ∼4 mM increase in subsarcolemmal [Na] compared to controls [7]. However, Baudet et al. [8] detected no change in intracellular Na activity in hypertrophied ferret heart using ISE. Data regarding [Na]i in heart failure are more limited. In an arrhythmogenic non-ischemic HF rabbit model (induced by aortic insufficiency and … * Corresponding author.

Frequency dependence of Ca2+ release from the sarcoplasmic reticulum in human ventricular myocytes from end-stage heart failure

OBJECTIVES Human cardiac muscle from failing heart shows a decrease in active tension development and a rise in diastolic tension at stimulation frequencies above 50-60 beats/min due to both systolic and diastolic dysfunction. We have investigated underlying changes in cellular [Ca2+]i regulation. METHODS Single ventricular myocytes were isolated enzymatically from the explanted hearts of transplant recipients with ischemic cardiomyopathy (nhearts = 5 ncells = 15) or dilated cardiomyopathy (nhearts = 6, ncells = 19). Cells were studied during whole-cell patch clamp with fluo-3 and fura-red as [Ca2+]i indicators (36 +/- 1 degrees C). RESULTS In current clamp mode (action potential recording), the amplitude of Ca2+ release from the sarcoplasmic reticulum (SR) decreased at stimulation frequencies above 0.5 Hz; this decrease was more pronounced for cells from dilated cardiomyopathy. Diastolic [Ca2+]i increased at 1 and 2 Hz for both groups. Action potential duration (APD90) decreased with frequency in all cells; in addition there was a drop in plateau potential of 10 +/- 1 mV for cells from ischemic cardiomyopathy and of 13 +/- 2 mV for cells from dilated cardiomyopathy. In voltage clamp mode the L-type Ca2+ current showed reversible decrease during stimulation at 1 and 2 Hz. Recovery from inactivation during a double pulse protocol was slow (75 +/- 3% at 500 ms, 89 +/- 3% at 1000 ms) and followed the decay of the [Ca2+]i transient. CONCLUSIONS The negative force-frequency relation of the failing human heart is due to a decrease in Ca2+ release of the cardiac myocytes at frequencies > or = 0.5 Hz, more pronounced in dilated than in ischemic cardiomyopathy. Inhibition of ICaL at higher frequencies, at least partially related to an increase in diastolic [Ca2+]i, will contribute to this negative staircase because of a decrease in the trigger for Ca2+ release, and of decreased loading of the SR.

Increased Na+ concentration and altered Na/K pump activity in hypertrophied canine ventricular cells

OBJECTIVE To investigate whether hypertrophy in the dog with chronic atrioventricular block (CAVB) alters [Na+]i and Na/K-pump function of ventricular myocytes. METHODS We measured the [Na+]i dependence of the Na/K pump current, I(p). This relation was used as a calibration curve for [Na+]i based on I(p). We measured I(p) at the time of access and extrapolated [Na+] at the pump sites, i.e. subsarcolemmal [Na+], [Na+](subs), from the calibration curve. RESULTS The extrapolated [Na+](subs) was significantly higher in CAVB (7.9 vs. 3.2 mM in control). The [Na+]i dependence of I(p) in CAVB myocytes was shifted to the right (range of [Na+](i): 0-20 mM). In resting cells, the I(p), i.e. steady state Na+ efflux, which matches Na+ influx, was higher in CAVB (0.25+/-0.02 vs. 0.47+/-0.06 pA/pF, P<0.05). Maximal I(p) density was not different, and DHO sensitivity was not altered. CONCLUSIONS Hypertrophy in CAVB cells is associated with increased [Na+](subs). This results from an increase in Na+ influx, and a decreased sensitivity of I(p) for Na+ in the range of [Na+]i studied. There is no evidence for a decrease in total pump capacity or for a functional Na/K-ATPase isoform shift. The rise in Na+ contributes to the contractile adaptation and preservation of sarcoplasmic reticulum Ca2+ content at the low heart rates of the dog with CAVB.

Intracellular Na+ and altered Na+ transport mechanisms in cardiac hypertrophy and failure

Altered intracellular Na(+) ([Na(+)](i)) is a potentially important factor in the functional adaptation of the hypertrophied and failing heart. We review the currently reported changes in [Na(+)](i) and Na(+) transport in different models of cardiac hypertrophy and heart failure. Direct measurements are limited, but most of these indicate that there is a rise in [Na(+)](i), in particular in hypertrophy. In addition to these direct measurements, several studies report a rise in Na(+) influx or an upregulation of Na(+) influx transporters. The most extensive literature on Na(+) regulating pathways concerns the Na/K-ATPase. Total Na/K-ATPase activity decreases in most models of cardiac hypertrophy and failure, though few measurements were actually performed in intact cells. This decrease can been related to a selective reduction of high-affinity (for cardiac glycosides) Na/K pump alpha-isoforms, across many species and models, including human heart failure. We have used these data to predict changes of [Na(+)](i) in a simulation model, varying the contribution of total Na/K pump capacity and expression of isoforms with different Na(+)(i) affinities, and varying Na(+) influx. A rise in Na(+) in cardiac hypertrophy and failure may improve systolic contractile function, though at the cost of worsening of diastolic function and increased risk of ventricular arrhythmias. The benefit of further increasing [Na(+)](i,) e.g. with cardiac glycosides, is thus compromised. Future therapies may include selective isoform blockers, which could raise [Na(+)](i) in restricted subcellular compartments, drug associations that reduce the arrhythmic risk, or even drugs that lower [Na(+)](i) and thus interfere with the remodelling pathways.

Electrophysiological effects of aprindine on isolated heart preparations.

Abstract The electrophysiological effects of aprindine, a new anti-arrhythmic drug, were investigated in isolated cardiac tissues of different animals. Diastolic depolarization and spontaneous firing were attenuated or abolished by aprindine. The action potential duration and effective refractory period were markedly shortened in Purkinje fibres. At the same concentrations, the action potential duration in atrial and ventricular muscle was not significantly altered. Membrane responsiveness curve was shifted to a more negative membrane potential. At concentrations above 5 mg/l, Purkinje fibres became inexcitable. Uptake and release of 14C-aprindine was studied in isolated heart preparations. Equilibration was not reached after an incubation period of 60 min and a final concentration of 10 times the concentration in the uptake fluid was attained after 60 min. The long-lasting effects on transmembrane potentials could be explained by the slow release of 14C-aprindine. A large fraction of the 14C-aprindine content was released with a time constant of 3 hr. The mechanism responsible for the in vivo anti-arrhythmic action and side effects are discussed.

Preferential block of the veratridine-induced, non-inactivating Na+ current by R56865 in single cardiac Purkinje cells

The effect of the cardioprotective agent R56865 on the veratridine (VTD)-modified sodium current was investigated in single rabbit cardiac Purkinje cells and ventricular myocytes. A steady, tetrodotoxin (TTX)-sensitive Na+ current (the non-inactivating Na+ current) was absent in most cells studied. In the presence of veratridine (15 x 10(-6) M) a non-inactivating Na+ current could be elicited at membrane potentials between -80 to +60 mV, with a maximum at about 0 mV. R56865 blocked this current effectively. The concentration for half maximal inhibition of the non-inactivating Na+ current was 2 x 10(-7) M. Blockade of this Na+ current by R56865 increased with depolarization. R56865 was much more effective in inhibiting the non-inactivating Na+ current than in inhibiting time-dependent Na+ currents elicited by short depolarizing pulses. The blocking effect of R56865 on the steady state influx of Na+ may contribute to cardioprotection in depolarized cells and in cells with modified Na+ channels as may occur during ischemia and reperfusion.

Evolutionary trace analysis of scorpion toxins specific for K-channels†

Scorpion α‐K+ channel toxins are a large family of polypeptides with a similar structure but diverse pharmacological activities. Despite many structural and functional data available at present, little progress has been made in understanding the toxin′s molecular basis responsible for the functional diversification. In this paper, we report the first complete cDNA sequences of toxins belonging to subfamily 6 and identify five new members, called α‐KTx 6.6‐6.10. By analyzing the rates of mutations that occurred in the corresponding cDNAs, we suggest that accelerated evolution in toxin‐coding regions may be associated with the functional diversification of this subfamily. To pinpoint sites probably involved in the functional diversity of α‐KTx family, we analyzed this family of sequences using the evolutionary trace method. This analysis highlighted one channel‐binding surface common for all the members. This surface is composed of one conserved lysine residue at position 29 assisted by other residues at positions 10, 26, 27, 32, 34, and 36. Of them, the positions 29, 32, and 34 have been reported to be the most major determinants of channel specificity. Interestingly, another contrary surface was also observed at a higher evolutionary time cut‐off value, which may be involved in the binding of ERG (ether‐a‐go‐go‐related gene) channel‐specific toxins. The good match between the trace residues and the functional epitopes of the toxins suggested that the evolutionary trace results reported here can be applied to predict channel‐binding sites of the toxins. Because, the side‐chain variation in the trace positions is strongly linked with the functional alteration and channel‐binding surface transfer of α‐KTx family, we conclude that our findings should also be important for the rational design of new toxins targeting a given potassium channel with high selectivity. Proteins 2004;54:000–000.