François Gannier

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 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.

Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular myocytes.

The effect of Gd3+ on the delayed rectifier potassium current (IK) in single guinea‐pig ventricular myocytes was tested using whole‐cell patch‐clamp techniques. It was found that Gd3+ blocked 70% of the IK tail current at a concentration of 100 microM. The EC50 was 24 microM. Action potential durations were, however, reduced, consistent with a predominant effect on depolarizing L‐type Ca2+ current (Ica.L). In the presence of 5 microM nifedipine Gd3+ prolonged the action potential. Using carbon fibres to stretch cells we observed that 10 microM Gd3+ was not effective in reducing a large stretch‐activated increase in resting calcium. Modelling studies using the OXSOFT HEART program suggest that this lack of response is influenced by blockade of repolarizing current but is best reproduced by additional blockade of Ca2+ extrusion via the Na(+)ࢤCa2+ exchanger. When Gd3+ is used as a blocker of stretch‐activated channels its actions upon both Ica.L and IK must therefore be accounted for.

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.

Antimony-induced cardiomyopathy in guinea-pig and protection by L-carnitine

1 Antimony (Sb) is the mainstay for the treatment of Leishmaniasis. It has serious, often lethal, cardiovascular side effects. The objective of this study was to examine the effects of Sb treatment upon the electrocardiogram (ECG), myocyte contractility (assessed by monitoring sarcomere length during field stimulation), whole‐cell action potential (AP) and calcium current (ICa) of the guinea‐pig and to evaluate L‐carnitine as a cardioprotective agent. 2 Guinea‐pigs received daily injections of either saline, Sb(V), Sb(III), L‐carnitine or L‐carnitine with Sb(III). Eight lead ECGs were recorded under halothane anaesthesia every 4 days. At the end of each treatment regime, animals were killed and ventricular myocytes were enzymatically isolated. 3 Treatment with Sb(V) for 26 days prolonged the QT interval of the ECG. Treatment with Sb(III) was lethal within 2 days for ∼50% of the animals. The survivors showed ECG alterations similar to those described in man: T wave flattening and/or inversion, depression of the ST segment, and elongation of RR and QT intervals. Their ventricular myocytes showed impaired contraction responses to changes in stimulus frequency, elongated AP and reduced ICa. 4 Combined treatment with L‐carnitine and Sb(III) delayed mortality. Prior treatment with L‐carnitine followed by combined treatment with L‐carnitine and Sb(III) reduced mortality to <10% over 12 days and these animals showed normal ECG. Their myocytes showed normal contractility and AP. 5 It is concluded that L‐carnitine has a preventive cardioprotective role against antimony‐induced cardiomyopathy. The mechanism of action of L‐carnitine may be to counter oxidative stress caused by Sb(III).

Altered heart rate control in transgenic mice carrying the KCNJ6 gene of the human chromosome 21

Congenital heart defects (CHD) are common in Down syndrome (DS, trisomy 21). Recently, cardiac sympathetic-parasympathetic imbalance has also been documented in DS adults free of any CHD. The KCNJ6 gene located on human chromosome 21 encodes for the Kir3.2/GIRK2 protein subunits of G protein-regulated K(+) (K(G)) channels and could contribute to this altered cardiac regulation. To elucidate the role of its overexpression, we used homozygous transgenic (Tg(+/+)) mice carrying copies of human KCNJ6. These mice showed human Kir3.2 mRNA expression in the heart and a 2.5-fold increased translation in the atria. Phenotypic alterations were assessed by recording electrocardiogram of urethane anesthetized mice. Chronotropic responses to direct (carbachol) and indirect (methoxamine) muscarinic stimulation were enhanced in Tg(+/+) mice with respect to wild-type (WT) mice. Alternating periods of slow and fast rhythm induced by CCPA (2-chloro-N-cyclopentyl-adenosine) were amplified in Tg(+/+) mice, resulting in a reduced negative chronotropic effect. These drugs reduced the atrial P wave amplitude and area. P wave variations induced by methoxamine and CCPA were respectively increased and reduced in the Tg(+/+) mice, while PR interval and ventricular wave showed no difference between Tg(+/+) and WT. These results indicate that Tg(+/+) mice incorporating the human KCNJ6 exhibit altered Kir3.2 expression and responses to drugs that would activate K(G) channels. Moreover, these altered expression and responses are limited to sino-atrial node and atria that normally express large amounts of K(G) channels. These data suggest that KCNJ6 could play an important role in altered cardiac regulation in DS patients.

SarcOptiM for ImageJ: high frequency online sarcomere length computing on stimulated cardiomyocytes

Accurate measurement of cardiomyocyte contraction is a critical issue for scientists working on cardiac physiology and physiopathology of diseases implying contraction impairment. Cardiomyocytes contraction can be quantified by measuring sarcomere length, but few tools are available for this, and none is freely distributed. We developed a plug-in (SarcOptiM) for the ImageJ/Fiji image analysis platform developed by the National Institutes of Health. SarcOptiM computes sarcomere length via fast Fourier transform analysis of video frames captured or displayed in ImageJ and thus is not tied to a dedicated video camera. It can work in real time or offline, the latter overcoming rotating motion or displacement-related artifacts. SarcOptiM includes a simulator and video generator of cardiomyocyte contraction. Acquisition parameters, such as pixel size and camera frame rate, were tested with both experimental recordings of rat ventricular cardiomyocytes and synthetic videos. It is freely distributed, and its source code is available. It works under Windows, Mac, or Linux operating systems. The camera speed is the limiting factor, since the algorithm can compute online sarcomere shortening at frame rates >10 kHz. In conclusion, SarcOptiM is a free and validated user-friendly tool for studying cardiomyocyte contraction in all species, including human.

Caffeine‐induced immobilization of gating charges in isolated guinea‐pig ventricular heart cells

The effects of 10 mM caffeine (CAF) on intramembrane charge movements (ICM) were studied in isolated guinea‐pig ventricular heart cells with the whole‐cell patch‐clamp technique. In the presence of CAF, the properties (voltage dependence, maximum QON[Qmax], availability with voltage) of QON charge activated from −110 mV were barely affected. Following a 100 ms prepulse to −50 mV to decrease the participation of charges originating from Na channels, the voltage dependence of QON was shifted by 5 mV (negative component) and by 10 mV (positive component) towards negative potentials, and Qmax was depressed by 16.5%. CAF drastically reduced in a time‐ and voltage‐dependent manner QOFF on repolarization to −50 mV, the effects being greater at positive potentials. CAF‐induced QOFF immobilization could be almost entirely removed by repolarization to voltages as negative as −170 mV. In these conditions, the voltage‐dependence of QOFF (repolarization to +30 to −170 mV) was shifted by 17 mV (negative component) and 30 mV (positive component) towards negative potentials, suggesting an interconversion into charge 2. Most of CAF effects were suppressed when the sarcoplasmic reticulum (SR) was not functional or when the cells were loaded with BAPTA‐AM. We conclude that CAF effects on ICM are likely due to Ca2+ ions released from the SR, and which accumulate in the subsarcolemmal fuzzy spaces in the vicinity of the Ca channels. Because CAF effects were more pronounced on QOFF than on QON the channels have likely to open before Ca2+ ions could affect their gating properties. It is speculated that such an effect on gating charges might contribute to the Ca‐induced inactivation of the Ca current.