Didier Garnier

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.

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.

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.

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.

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.

Orthophosphate salts induce calcium current recovery from blockade by gadolinium in isolated guinea-pig ventricular myocytes.

Using the whole‐cell configuration of the patch‐clamp technique, we studied the incomplete recovery of the high voltage‐activated calcium current (ICa,L) from a complete blockade by 10 microM gadolinium. The study was performed on isolated guinea‐pig ventricular myocytes. ICa,L recovery depended on the duration of application of Gd3+: for a mean application time of 1.63 +/− 0.45 min, a recovery to 67 +/− 22% of the initial current amplitude (n = 12 cells) was observed; when the application lasted 5.1 +/− 1.2 min ICa,L recovered to 27 +/− 13% (n = 14 cells). The partial recovery of ICa,L was accompanied by a slowing of the inactivation phase of the current. IF, during the incomplete recovery, cells were exposed to a solution containing 330 microM of an orthophosphate salt, such as NaH2PO4 or KH2PO4, the current amplitude increased to 80 +/− 13% of the initial current (n = 10 cells) in a reversible manner. However, the slowing of the inactivation phase was maintained. Our results show that the partial recovery of ICa,L from blockade by gadolinium is due to an interaction of the blocker with an extracellular part of the channel, possibly one involved in voltage‐dependent inactivation.

SEVERE AND EARLY ALTERATION OF ACTION POTENTIAL DURING ACUTE CARDIAC REJECTION IN RATS

Alteration of Cardiac Action Potential. Introduction: Alteration of cardiac action potential and its adaptation to heart rate could contribute to cardiac dysfunction and arrhythmias during acute cardiac rejection.

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 (> 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.