Jorge Argibay

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.

The effects of increasing cell length on auxotonic contractions; membrane potential and intracellular calcium transients in single guinea‐pig ventricular myocytes

Until recently the investigation of length‐dependent effects in cardiac muscle was restricted to multicellular preparations. We describe our experimental set‐up which for the first time, in single cardiac myocytes, permits the effects of changes in cell length on auxotonic contractions (measured by carbon fibre transducers) to be simultaneously recorded with the effects on membrane potential and/or changes in intracellular calcium concentration (using indo‐1 AM, acetoxylmethyl form). Consistent with previous findings (in experiments at 20‐25 degrees C and 0.25 Hz) we report that following a stretch there was an increase in passive tension and contraction. A stretch which increased sarcomere length by approximately 3% had no significant effect on resting membrane potential or action potential amplitude. There was, however, a significant decrease in the action potential duration (P < 0.01, n = 8). No significant change in the amplitude of the intracellular calcium transient was seen following a stretch but a reduction in its duration was observed (P < 0.025, n = 11). Our observations on intracellular calcium transients are consistent with the hypothesis that, in mechanically loaded preparations, their time course is more dependent on changes in tension than changes in length.

Anti-SSA/Ro52 autoantibodies blocking the cardiac 5-HT4 serotoninergic receptor could explain neonatal lupus congenital heart block

The 52‐kDa SSA/Ro (Ro52) ribonucleoprotein is an antigenic target strongly associated with the autoimmune response in mothers whose children develop neonatal lupus and congenital heart block. When sera from patients with systemic lupus erythematosus were used as autoimmune controls in an enzyme immunoassay to screen for antibodies against the human serotoninergic 5‐HT4‐receptor, a high correlation was found between the presence of anti‐Ro52 protein antibodies in such sera and antibodies reacting with a synthetic peptide, corresponding to the second extracellular loop of the human 5‐HT4 receptor (amino acid residues 165–185). Homology scanning between the 5‐HT4 peptide and the sequence of the Ro52 protein indicated two potential common epitopes located between residues 365 and 396 of the Ro52 protein. Cross‐reactivity was found between the peptide derived from the 5‐HT4 receptor, and a peptide corresponding to residues 365–382 of the Ro52 protein. Autoantibodies, affinity‐purified on the 5‐HT4 receptor peptide, specifically recognized both the Ro52 protein and the 5‐HT4 receptor protein in immunoblots. The affinity‐purified antibodies antagonized the serotonin‐induced L‐type Ca channel activation on human atrial cells. This effect could explain the electrophysiological abnormalities in neonatal lupus.

A NEW METHOD OF ATTACHMENT OF ISOLATED MAMMALIAN VENTRICULAR MYOCYTES FOR TENSION RECORDING : LENGTH DEPENDENCE OF PASSIVE AND ACTIVE TENSION

The study of the Frank-Starlings law in mammalian single cells has been hindered by a lack of an easily performed method of stretching cells. Some authors have succeeded in this but their methods required a great deal of technical expertise and in most cases they have not had much success. We have developed an easy method of stretching mammalian ventricular cells from slack sarcomere length (S.L.) (Lo, 1.77 +/- 0.05 microns) to about 117% of this length. Thin carbon fibers (12 microns in diameter) which can be bound electrochemically to the cell membrane surface have been used. A flexible long fiber of known compliance (80 microns/microN) was attached to one end of the cell and a stiff double fiber (4 microns/microN) to the other end. The cell attachment was relatively easy to perform and successful results were obtained in 80% of the attempts. The displacement of the flexible fiber allows the quantitative measurements of the resting tension in a group of non-stimulated cells and of auxotonic contractions developed upon stimulation in another group of cells. Increasing S.L. from Lo to 105-106% of Lo, an increase in active tension from 0.21 +/- 0.03 mN/mm to 0.26 +/- 0.01 mN/mm (n = 4) could be noticed with a stimulation frequency of 0.5 Hz. An increase in active tension was also observed at 1 Hz. Staircase kinetics were accelerated with stretching; this confirms at the single cell level the hypothesis of an effect of length-dependent activation on the staircase. Eulerian differential stiffness constant was calculated and found to be 13.5 +/- 1.2, a value which is comparable to that described in intact heart. Thus the important stiffness found in the whole heart may be due to intracellular component(s) such as myofilament and/or connectin.

Stretch‐induced increase of resting intracellular calcium concentration in single guinea‐pig ventricular myocytes

Single guinea‐pig ventricular myocytes were loaded with the fluorescent Ca2+ indicator Indo‐1 AM and stretched by carbon fibres. Stretching increased resting tension. Sarcomere lengths were increased by 2‐18%. It was observed that a stretch increased resting [Ca2+]i in seven out of eight cells. The change in [Ca2+]i increased with the size of the stretch and returned to pre‐stretch levels on return to resting cell length. These observations suggest a means by which changes in resting muscle length can modify the contractile state of cardiac muscle.

Anti-peptide antibodies sensitive to the ‘active’ state of the β2-adrenergic receptor

Antibodies directed against a peptide corresponding to the second loop of the human β 2‐adrenergic receptor were induced in rabbits by immunisation with the free peptide in complete Freunds adjuvant. The resulting antibodies were affinity‐purified and shown to be monospecific for the target receptor. They were able to stimulate the L‐type Ca2+ channels in whole‐cell patch‐clamp experiments on isolated adult guineapig cardiomyocytes. This effect was similar to that obtained by the specific β 2‐adrenergic agonist zinterol. The antibody effects could be blocked with the specific β 2‐adrenergic inverse agonist ICI118,551 but not with the neutral antagonist alprenolol. These results suggest that the antibodies recognise the active conformer of the β 2‐adrenergic receptor.

A simple method for calibrating collagenase/pronase E ratio to optimize heart cell isolation

Summary— A mixture of crude collagenase and non‐specific proteases has been used to isolate guinea pig ventricular heart cells. Measurements of collagenase activity with Wünschs substrate and protein content with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) suggest that collagenase enzymes do not play a major role in heart cell isolation. On the other hand, an important factor in heart digestion seems to consist of some fractions of the proteases present in crude collagenase. It is also noted that crude collagenases do not present any sensitivity to added calcium but because this ion is important to obtain isolated cells its role is discussed. According to our results, the SDS‐PAGE method can be used to determine the appropriate enzyme concentrations to obtain calcium‐tolerant myocytes. These myocytes have electrophysiological properties as reported in the literature.

Rate dependence of action potential duration and calcium current in isolated guinea‐pig cardiocytes

The duration of the action potential at 50% of its amplitude (APD50) and peak calcium currents (ICa) was measured in single cardiac guinea‐pig ventricular cells, using the whole‐cell patch‐clamp technique in current‐clamp and voltage‐clamp modes respectively. In the absence of intracellular calcium buffer, pacing at 0.28 Hz from a rest period of 1‐2 min induced a transient increase (15.5 +/− 3.5%) in APD50, followed by a gradual shortening. Switching from 0.28 to 0.75 Hz again induced a transient increase of APD50 (6.8 +/− 2.9%). In the presence of EGTA or BAPTA on the cytosolic side of the membrane, this transient phase was prolonged and its amplitude slightly increased (10.6% when switching from 0.28 to 0.75 Hz in 5 mM‐BAPA). The same increase in rate induced either a negative or a positive staircase of ICa, depending on the holding potential. At a holding potential of ‐80 mV, ICa peak was enhanced and the inactivation kinetics was slowed down. This facilitation of ICa seems to be dependent on calcium ions entering the cell via the calcium channels and could partly explain the observed transient increase in APD50.

Ruthenium red as an effective blocker of calcium and sodium currents in guinea‐pig isolated ventricular heart cells

The effect of ruthenium red on calcium and sodium currents was studied in guinea‐pig isolated ventricular heart cells with the whole cell patch‐clamp technique. Ruthenium red very efficiently blocked the L‐type calcium current in a dose‐dependent manner. A significant block was observed for concentrations as low as 0.3 μM. Analysis of the dose‐response curve with the logistic equation indicated an EC50 of 0.8 μM, a maximum inhibition of 85% reached at 5 μM, and a coefficient of 2.37. There was no shift in the voltage‐dependence of the Ca current activation, nor in that of its steady‐state inactivation determined with a 1 s prepulse. However, removal of Ca current inactivation at positive voltage was considerably reduced in the presence of concentrations of ruthenium red above 1 μM. A slowing of the time‐course of inactivation of the Ca current was also observed. At 10 μM, a concentration generally used to block the sarcoplasmic Ca release channels or the mitochondrial Ca uptake, ruthenium red blocked 26.7±4.3% (n=8) of the sodium current, and slowed its inactivation time‐course. No effect was observed on the voltage‐dependence of the current activation or inactivation. The peak sodium current was also decreased at a 10 times lower concentration by 7.6±2.7% (n=3). Thus, at concentrations used to assess intracellular Ca movements, ruthenium red induced in heart cells a significant block of both Ca and Na channels.

Block of gating currents related to K+ channels as a mechanism of action of clofilium and d-sotalol in isolated guinea-pig ventricular heart cells

The possibility that the class III antiarrhythmic drugs clofilium and d‐sotalol might affect delayed rectifier potassium channels at the level of their gating currents was assessed with the whole‐cell patch‐clamp technique in guinea‐pig isolated ventricular heart cells. Clofilium (up to 20 μM) and d‐sotalol (1 μM) did not decrease the Na current, the L‐type Ca current or the background K current iKl, but significantly depressed the time‐dependent delayed outward K current iK. Clofilium partially decreased in a dose‐dependent manner (1–20 μM) QON of intramembrane charge movements (ICM) elicited by a depolarizing pulse applied from a holding potential of −110 mV or following a 100 ms inactivating prepulse to −50 mV. D‐sotalol (1 μM) also decreased QON. Channel density estimated from the clofilium‐sensitive ICM closely matched that of the delayed rectifier channels. Clofilium and d‐sotalol decreased QOFF seen on repolarization in a dose‐ and voltage‐dependent manner. The kinetics of the decay of the OFF gating currents were not affected, and only the fast phase was depressed. In control conditions, QON availability with voltage was most of the time well described by two inactivating components. In the presence of clofilium and d‐sotalol, a complex behaviour of QON availability was observed, unmasking additional components. The reactivation kinetics of QON after a 500 ms inactivating pulse to 0 mV was not affected. We conclude that delayed rectifier K channels significantly contribute to QON and QOFF of ICM in guinea‐pig ventricular heart cells, besides Na and Ca channels, and that clofilium and d‐sotalol directly interact with these K channels proteins by affecting their gating properties.