Palmitoyl-carnitine increases RyR2 oxidation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes: Role of adenine nucleotide translocase.

Long chain fatty acids bind to carnitine and form long chain acyl carnitine (LCAC), to enter into the mitochondria. They are oxidized in the mitochondrial matrix. LCAC accumulates rapidly under metabolic disorders, such as acute cardiac ischemia, chronic heart failure or diabetic cardiomyopathy. LCAC accumulation is associated with severe cardiac arrhythmia including ventricular tachycardia or fibrillation. We thus hypothesized that palmitoyl-carnitine (PC), alters mitochondrial function leading to Ca(2+) dependent-arrhythmia. In isolated cardiac mitochondria from C57Bl/6 mice, application of 10µM PC decreased adenine nucleotide translocase (ANT) activity without affecting mitochondrial permeability transition pore (mPTP) opening. Mitochondrial reactive oxygen species (ROS) production, measured with MitoSOX Red dye in isolated ventricular cardiomyocytes, increased significantly under PC application. Inhibition of ANT by bongkrekic acid (20 µM) prevented PC-induced mitochondrial ROS production. In addition, PC increased type 2 ryanodine receptor (RyR2) oxidation, S-nitrosylation and dissociation of FKBP12.6 from RyR2, and therefore increased sarcoplasmic reticulum (SR) Ca(2+) leak. ANT inhibition or anti-oxidant strategy (N-acetylcysteine) prevented SR Ca(2+) leak, FKBP12.6 depletion and RyR2 oxidation/S-nitrosylation induced by PC. Finally, both bongkrekic acid and NAC significantly reduced spontaneous Ca(2+) wave occurrences under PC. Altogether, these results suggest that an elevation of PC disturbs ANT activity and alters Ca(2+) handling in a ROS-dependent pathway, demonstrating a new pathway whereby altered FA metabolism may contribute to the development of ventricular arrhythmia in pathophysiological conditions.

Effect of ultrasound-activated microbubbles on the cell electrophysiological properties.

New clinical applications of ultrasound contrast microbubbles extend beyond imaging and diagnosis toward therapeutic applications. Cell membrane permeability and the uptake of substances have been shown to be enhanced by microbubbles under ultrasound stimulation. However, the mechanisms of action of ultrasound-activated microbubbles are still unknown. The aim of our study was to examine how microbubbles and ultrasound interact with cells in an attempt to understand the sonoporation mechanism. The ruptured-patch-clamp whole-cell technique was used to measure membrane potential variations of a single cell. SonoVue microbubbles and mammary breast cancer cell line MDA-MB-231 were used. Ultrasound was applied using single-element transducers of 1 MHz. Microbubbles and cells were simultaneously video monitored during ultrasound exposure. Our results showed that, during sonoporation, a marked cell membrane hyperpolarization occurs (n = 6 cells) at negative pressures above 150 kPa, indicating the activation of specific ion channels while the cell and the microbubbles remain viable. The hyperpolarization was sustained for as long as the microbubbles are in a direct contact with the cell and the ultrasound waves are transmitted. Smaller acoustic amplitudes induced only mild hyperpolarization, whereas shutting off the ultrasound brings the cell membrane potential to its resting value. However, ultrasound alone did not affect the cell membrane potential. A similar hyperpolarization of the cell membrane was observed when a mechanical pressure was applied on the cell through a glass probe. In conclusion, the results demonstrate that microbubbles' oscillations under ultrasound activation entail modifications of the electrophysiologic cell activities by triggering the modulation of ionic transports through the plasmic cell membrane. However, only cells in direct contact with the microbubbles are impacted. The mechanisms involved are likely related to activation of specific channels sensitive to mechanical stresses (stretch-activated channels) and possibly nonspecific ion channels.

Modulation of Ca(2+) signaling by microtubule disruption in rat ventricular myocytes and its dependence on the ruptured patch-clamp configuration.

In the absence of hypertrophic proliferation of microtubules, microtubule disruption by colchicine does not modulate contraction of adult cardiac myocytes. However, Gomez et al (Circ Res. 2000;86:30-36) recently reported that disruption of microtubules by colchicine in ruptured patch-clamped myocytes increased I(Ca,L) density and [Ca(2+)](i) transient amplitude and depressed the response of these parameters to the beta-adrenoceptor agonist isoproterenol. These effects were ascribed to stimulation of adenylyl cyclase by increased intracellular free tubulin. In the present study, we show that in intact rat ventricular myocytes, 2 to 4 hours of exposure to 10 micromol/L colchicine had no effect on shortening or [Ca(2+)](i) transient amplitude or on the amplitude of I(Ca,L) in perforated patch-clamped cells, under basal conditions and after stimulation with 1 micromol/L isoproterenol. However, in ruptured patch-clamped myocytes, basal I(Ca,L) was 2-fold higher after treatment with colchicine compared with vehicle and, in contrast to vehicle-treated cells, I(Ca,L) did not increase in response to isoproterenol. Cell width decreased during ruptured patch-clamp experiments in colchicine-treated but not vehicle-treated myocytes. We conclude that in cells with intact sarcolemma, colchicine does not modulate Ca(2+) signaling or the response to beta stimulation. However, the combination of microtubule disruption by colchicine and the ruptured patch configuration activates I(Ca,L) and attenuates the response to beta stimulation. We propose that these effects may be due to loss of free tubulin by intracellular dialysis or to increased sensitivity to mechanical stimulation as a result of microtubule disruption. These findings have important implications for cardiomyopathies associated with decreased free tubulin or a diminished microtubular network. The full text of this article is available at http://www.circresaha.org.

The high frequency relationship: implications for torsadogenic hERG blockers.

BACKGROUND AND PURPOSE: Ventricular arrhythmias induced by human ether-a-go-go related gene (hERG; Kv 11.1 channel) blockers are a consequence of alterations in ventricular repolarisation in association with high-frequency (HF) oscillations, which act as a primary trigger; the autonomic nervous system plays a modulatory role. In the present study, we investigated the role of ß1 -adrenoceptors in the HF relationship between magnitude of heart rate and QT interval changes within discrete 10 s intervals (sorted into 5 bpm heart rate increments) and its implications for torsadogenic hERG blockers. EXPERIMENTAL APPROACH: The HF relationship was studied under conditions of autonomic blockade with atenolol (ß1 -adrenoceptor blocker) in the absence or presence of five hERG blockers in beagle dogs. In total, the effects of 14 hERG blockers on the HF relationship were investigated. KEY RESULTS: All the torsadogenic hERG blockers tested caused a vertical shift in the HF relationship, while hERG blockers associated with a low risk of Torsades de Pointes did not cause any vertical shift. Atenolol completely prevented the effects four torsadogenic agents (quinidine, thioridazine, risperidone and terfenadine) on the HF relationship, but only partially reduced those of dofetilide, leading to the characterization of two types of torsadogenic agent. CONCLUSIONS AND IMPLICATIONS: Analysis of the vertical shift in the HF relationship demonstrated that signs of transient sympathetic activation during HF oscillations in the presence of torsadogenic hERG blockers are mediated by ß1 -adrenoceptors. We suggest the HF relationship as a new biomarker for assessing Torsades de pointes liability, with potential implications in both preclinical studies and the clinic.

The stretch-activated ion channel blocker gadolinium also blocks L-type calcium channels in isolated ventricular myocytes of the guinea-pig.

We show that gadolinium (Gd3+) is a potent calcium channel blocker in guinea-pig isolated ventricular myocytes. A dose-dependent inhibition of ICaL was found with an EC50 of 1.4 microM and a complete inhibition at 10 microM Gd3+. When compared with Cd2+, it appeared that the blockade of ICaL is a complex phenomenon probably involving more than one site of interaction (a Hill coefficient of 1.6 was found for Gd3+ vs. 1.0 for Cd2+). It is concluded that Gd3+ ions completely block ICaL at concentrations used to block stretch-activated channels (SAC), rendering its use as a specific SAC inhibitor problematic.

Cytoskeletal modulation of electrical and mechanical activity in cardiac myocytes.

The cardiac myocyte has an intracellular scaffold, the cytoskeleton, which has been implicated in several cardiac pathologies including hypertrophy and failure. In this review we describe the role that the cytoskeleton plays in modulating both the electrical activity (through ion channels and exchangers) and mechanical (or contractile) activity of the adult heart. We focus on the 3 components of the cytoskeleton, actin microfilaments, microtubules, and desmin filaments. The limited visual data available suggest that the subsarcolemmal actin cytoskeleton is sparse in the adult myocyte. Selective disruption of cytoskeletal actin by pharmacological tools has yet to be verified in the adult cell, yet evidence exists for modulation of several ionic currents, including I(CaL), I(Na), I(KATP), I(SAC) by actin microfilaments. Microtubules exist as a dense network throughout the adult cardiac cell, and their structure, architecture, kinetics and pharmacological manipulation are well described. Both polymerised and free tubulin are functionally significant. Microtubule proliferation reduces contraction by impeding sarcomeric motion; modulation of sarcoplasmic reticulum Ca(2+) release may also be involved in this effect. The lack of effect of microtubule disruption on cardiac contractility in adult myocytes, and the concentration-dependent modulation of the rate of contraction by the disruptor nocodazole in neonatal myocytes, support the existence of functionally distinct microtubule populations. We address the controversy regarding the stimulation of the beta-adrenergic signalling pathway by free tubulin. Work with mice lacking desmin has demonstrated the importance of intermediate filaments to normal cardiac function, but the precise role that desmin plays in the electrical and mechanical activity of cardiac muscle has yet to be determined.

A possible mechanism for large stretch-induced increase in [Ca2+]i in isolated guinea-pig ventricular myocytes.

OBJECTIVES: The aim of the study was to investigate the mechanisms responsible for provoking and maintaining a large, stretch-induced, increase in the level of resting calcium in single guinea-pig ventricular myocytes. In particular, we wished to test the relative importance of intracellular and extracellular sources of calcium in this phenomenon. METHODS: Carbon fibres were used to stretch cells loaded with the fluorescent calcium indicator Indo-1. Sarcomere length and internal calcium activity ([Ca2+]i) were measured. Experimental results from our present and previous studies were compared with those predicted by the OXSOFT HEART (version 4) model of the guinea-pig ventricular myocyte incorporating a stretch-activated channel. RESULTS: The stretch-induced increase in [Ca2+]i was found to be sensitive to removal of [Ca2+]o and application of the Ca(2+)-channel blocker verapamil (1 microM). The phenomenon was not sensitive to disruption of sarcoplasmic reticulum function by ryanodine (1 microM) nor to the Na+ channel blocker TTX (30 microM). Our experimental findings were reproduced in the modelling study. CONCLUSIONS: The stretch-induced increase in [Ca2+]i is modulated by extracellular sources of Ca2+ rather than intracellular Ca2+ stores and is not indiscriminately sensitive to blockers of depolarizing current. We propose that the stretch-induced increase in [Ca2+]i may be triggered by activation of stretch-activated channels but that a combination of stretch-activated current and Ca(2+)-window current maintain the increased levels of resting [Ca2+]i.

Streptomycin reverses a large stretch induced increases in [Ca2+]i in isolated guinea pig ventricular myocytes.

OBJECTIVE: The aim was to test the hypothesis that in single guinea pig ventricular myocytes a large stretch induced increase in resting calcium was sensitive to the mechanosensitive channel blocker streptomycin. METHODS: Carbon fibres were used to stretch cells loaded with the fluorescent calcium indicator indo-1. Force, sarcomere length, and internal calcium activity ([Ca2+]i) were measured. RESULTS: In approximately 60% of the cells studied, a stretch which increased sarcomere length by approximately 6% caused a large increase in [Ca2+]i (up to 60% of the size of a [Ca2+]i transient at 0.25 Hz). When a mixture of antibiotics (streptomycin-penicillin) was used in solutions to isolate and store cells, this phenomenon was never observed (n = 19 cells). Direct application of physiological saline solution (PSS) could not reverse the increase in [Ca2+]i within 60 s of application (n = 7 cells). Direct application of penicillin [1000 IU per 50 ml (40 microM)] reversed the increase in [Ca2+]i within 60 s of application in only 3/7 cells. In contrast direct application of the aminoglycoside antibiotic streptomycin (40 microM) rapidly reversed the large increase in [Ca2+]i induced by stretch in each of 13 cells [within 18(SD 10) s of application]. Acute application of 40 microM streptomycin did not modify L-type Ca2+ currents measured under whole cell patch clamp conditions. Measurement of the resting tension--sarcomere length curves in cells stored in solution containing streptomycin and penicillin revealed two populations of cells on the basis of their stiffness. CONCLUSIONS: This stretch induced increase in [Ca2+]i may be associated with stretch activated arrhythmias in the heart. The effects of streptomycin are consistent with its reported inhibitory action on stretch activated channels.

Short-term variability in QT interval and ventricular arrhythmias induced by dofetilide are dependent on high-frequency autonomic oscillations.

BACKGROUND AND PURPOSE: The present study was undertaken to investigate an effect of dofetilide, a potent arrhythmic blocker of the voltage-gated K(+) channel, hERG, on cardiac autonomic control. Combined with effects on ardiomyocytes, these properties could influence its arrhythmic potency. EXPERIMENTAL APPROACH: The short-term variability of beat-to-beat QT interval (STVQT ), induced by dofetilide is a strong surrogate of Torsades de pointes liability. Involvement of autonomic modulation in STVQT was investigated in healthy cynomolgus monkeys and beagle dogs by power spectral analysis under conditions of autonomic blockade with hexamethonium. KEY RESULTS: Increase in STVQT induced by dofetilide in monkeys and dogs was closely associated with an enhancement of endogenous heart rate and QT interval high-frequency (HF) oscillations. These effects were fully suppressed under conditions of autonomic blockade with hexamethonium. Ventricular arrhythmias, including Torsades de pointes in monkeys, were prevented in both species when HF oscillations were suppressed by autonomic blockade. Similar enhancements of heart rate HF oscillations were found in dogs with other hERG blockers described as causing Torsades de pointes in humans. CONCLUSIONS AND IMPLICATIONS: These results demonstrate for the first time that beat-to-beat ventricular repolarization variability and ventricular arrhythmias induced by dofetilide are dependent on endogenous HF autonomic oscillations in heart rate. When combined with evidence of hERG-blocking properties, enhancement of endogenous HF oscillations in heart rate could constitute an earlier and more sensitive biomarker than STVQT for Torsades de pointes liability, applicable to preclinical regulatory studies conducted in healthy animals.

An enzyme immunoassay design using labelled antibodies for the determination of haptens. Application to methotrexate assay.

A competitive enzyme immunoassay with labelled antibodies has been developed for methotrexate (MTX). Methotrexate in the sample and a constant quantity of this hapten physically absorbed to polystyrene spheres through a methylated bovine albumin carrier were allowed to compete for a limiting amount of peroxidase labelled antibody. After washing, the residual enzyme activity bound to the solid phase was measured. This test was able to detect 10 fm of MTX per sample. A comparative study of this test with a commercial radioimmunoassay kit using the same antiserum and a high pressure liquid chromatography method showed that the sensitivity, specificity and precision of this test were as good as of the radioimmunoassay. The high pressure liquid chromatography method was 500 times less sensitive. Good agreement was found among the 3 methods on 83 serum samples from patients receiving methotrexate therapy.

Alteration of the L-type calcium current in guinea-pig single ventricular myocytes by heptaminol hydrochloride.

1. The effects of heptaminol on calcium current amplitude and characteristics were studied in single ventricular myocytes of guinea-pig by use of the whole cell configuration of the patch clamp technique. 2. A concentration-dependent decrease in ICa amplitude was observed. At heptaminol concentration as low as 10(-6) M, this effect was observed in only two cells (n = 6). At 10(-5) M the reduction of ICa was of 30 +/- 15% (n = 11). 3. The current recovery from inactivation at -40 mV holding potential (HP) seemed less sensitive to perfusion with heptaminol (greater than 10(-6) M). However, at -80 mV HP the overshoot of the recovery curve was decreased by heptaminol. 4. Both at -40 mV and -80 mV HP, heptaminol (10(-5) M) significantly increased the steady state inactivation of ICa. 5. As previously proposed by others to explain the effects of membrane active substances, the effects of heptaminol may result from alterations in cell membrane properties and possibly from an increase in intracellular free calcium ion concentration.

Different effects of gadolinium on I(KR), I(KS) and I(K1) in guinea-pig isolated ventricular myocytes.

1. Using the whole cell configuration of the patch clamp technique, we studied the potential blocking effects of gadolinium (1 microM to 1 mM) on potassium currents: I(KR), I(KS) and I(K1). The study was performed on guinea-pig isolated ventricular myocytes. 2. The background current, I(K1) was insensitive to Gd3+. Thus, we found that no obvious screening of surface charges was visible with concentrations of Gd3+ up to 100 microM. 3. By use of three different protocols: tail currents fitting, analysis of envelope of tails and electrophysiological dissection, we found that I(KR) was the only component of IK that was sensitive to Gd3+. The sensitivity was apparently different depending on the protocol used. 4. Comparison of the results obtained with the different protocols revealed that the rapid component of I(KR) is more sensitive to Gd3+ than the slow one. 5. Of the different protocols used to distinguish between I(KR) and I(KS), the electrophysiological dissection seems to be the more accurate.

Peroxidation of docosahexaenoic acid is responsible for its effects on I TO and I SS in rat ventricular myocytes.

1 Exposure to docosahexaenoïc acid (DHA), a long-chain polyunsaturated fatty acid, is known to block several ionic currents such as the transient outward current I(TO). It has also been reported to activate certain potassium channels. It has been suggested that these effects, observed in single-cell experiments, participate in the antiarrhythmic properties of these compounds in vivo. 2 DHA is highly prone to peroxidation. To investigate the influence peroxidation may have on the effects of DHA on ion channels, we studied I(TO) and the steady-state outward current I(SS) in isolated rat ventricular myocytes under ruptured whole-cell patch-clamp conditions. 3 A measure of 10 micro M DHA alone reduced I(TO), evoked by a pulse to +70 mV, by 74.8+/-10.8% (n=7) and activated a delayed outward current with kinetic properties different from I(SS). 4 When an antioxidant, alpha-tocopherol (1 micro M), was added together with DHA, the blockade of I(TO) was reduced to 38.5+/-7.7% (n=8) and the delayed outward current was not activated. alpha-Tocopherol alone had no effect on these currents. 5 When an oxidant, hydrogen peroxide (1 micro M), was applied together with DHA, the blockade of I(TO) was almost complete (98.4+/-1.0%, n=7) and a large delayed outward current was activated. A measure of 1 micro M hydrogen peroxide alone had no effect on these currents. 6 Measurements of nonperoxidized DHA in experimental solutions confirmed the negative relation between DHA concentration and the effects on the currents. 7 We conclude that rather than DHA itself, it is the peroxidation products of DHA that block I(TO) and activate a delayed outward current in in vitro single-cell experiments. These findings have important implications for the extrapolation of in vitro experimental findings to the antiarrhythmic effects of DHA in vivo because, in vivo, peroxidation of DHA is unlikely to occur.

Antithrombin III synthesis in rat liver parenchymal cells.

We have previously demonstrated by immunoperoxydase the presence of immunoreactive antithrombin III (AT III) in rat hepatocytes. We now present direct evidence that rat hepatocytes in culture synthesize AT III like immunoreactive material : 35S-methionine was added to the culture medium and incubated with hepatocytes. After incubation, AT III was immunologically characterized in the medium. We found significant amounts of 35S-AT III among the radioactive proteins synthesized and secreted by the cells.

Is titin the length sensor in cardiac muscle? Physiological and physiopathological perspectives.

One of the most salient physiological characteristics of cardiac muscle is that a dilated heart pumps more vigorously, a phenomenon known as the Frank-Starling relationship (see Allen and Kentish, 1985). At least two cellular mechanisms participate in this phenomenon: the reduction of the interfilament lattice spacing which favors the formation of cross-bridges (Wang and Fuchs, 1995) and the increased affinity of troponin C (TnC) for calcium (Ca2+) (Babu et al., 1988). In the latter case, it has been established that TnC itself is not the length sensor (Moss et al., 1991). The intracellular structure(s) able to sense changes in cell length has always been challenged and is still not known. We previously observed on intact isolated cardiac cells that active tension is more closely related to passive tension than to sarcomere length per se (Cazorla et al., 1997). This might have some physiological implications in the working heart since we found that sub-epicardial cells are more supple than sub-endocardial cells. In the present work on skinned cells, we studied the relationship between different levels of passive tension (modulated by a mild trypsin digestion) and the shift in pCa50 of tension-pCa relations induced by a stretch of cells from 1.9 to 2.3 microns sarcomere length. A significant correlation was obtained between passive tension and the stretch-induced shift in pCa50, or stretch-sensitivity of the active force. These observations led us to assume that titin might play a role in sensing cell length to modulate the contractile activity. Besides, it is known that myocardial infarcted cells are less sensitive to stretch. We propose that, in such a rat model, alterations of titin might participate in heart failure.

Na(V)1.5 enhances breast cancer cell invasiveness by increasing NHE1-dependent H(+) efflux in caveolae.

Na(V)1.5 sodium channels enhance the invasiveness of breast cancer cells through the acidic-dependent activation of cysteine cathepsins. Here, we showed that the Na(+)/H(+) exchanger type 1 (NHE1) was an important regulator of H(+) efflux in breast cancer cells MDA-MB-231 and that its activity was increased by Na(V)1.5. Na(V)1.5 and NHE1 were colocalized in membrane rafts containing caveolin-1. The inhibition of Na(V)1.5 or NHE1 induced a similar reduction in cell invasiveness and extracellular matrix degradation; no additive effect was observed when they were simultaneously inhibited. Our study suggests that Na(V)1.5 and NHE1 are functionally coupled and enhance the invasiveness of cancer cells by increasing H(+) efflux.

Cardioprotection by omega-3 fatty acids: involvement of PKCs?

It has been known since the 1970s that an increased consumption of n-3 long chain polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid has cardioprotective effects. Epidemiological studies have reported that this effect is due to the prevention of the arrhythmias responsible for sudden cardiac death. Mechanistically, different hypotheses have been put forward to give an explanation. Among them, there are a direct effect of the polyunsaturated fatty acids on ion channels and/or a modification of the regulation of ion channels by protein kinase C's.