We examined by a statistical approach the decrease of the Ca current ("run-down") during long-lasting recordings with the whole-cell patch-clamp technique in guinea pig ventricular myocytes. The results are as follows. (1) Run-down of the Ca current (ICa) occurs in three phases (T1-T3). T1 (38 +/- 19 min, n = 135) and T3 (35 +/- 17 min, n = 23) are characterized by a slow rate of decay of ICa [90 +/- 20 and 60 +/- 20 nA.cm-2.min-1, respectively]. T1 and T3 are separated by T2 (6 +/- 4 min, n = 135) during which the current decays quickly [1200 +/- 230 nA.cm-2.min-1]. Between the onsets of T1 and T3, ICa decreases from 11 +/- 3 to 3.5 +/- 1 microA/cm2. (2) Normalized current-voltage relationship, reversal potential and voltage-dependencies of steady-state activation and inactivation of ICa are globally shifted toward more negative potentials during the run-down process by 10-15 mV. (3) ICa3 measured during T3 retains the pharmacological properties (blockade by D600, NiCl2 and CoCl3, increase by isoprenaline and insensitivity to tetrodotoxin) of the original ICa. (4) Intracellular perfusion of the nonhydrolysable ATP analogue AMP-PNP does not prevent the occurrence of T2, suggesting that a phosphorylation-dephosphorylation process is not involved in the fast run-down of ICa. (5) With 0.1 mM EGTA in the pipette, addition of 3 mM ATP significantly prolongs ICa survival. No improvements are obtained by increasing the ATP concentration to 10 mM or replacing ATP with creatine phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

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Ca2+-dependent regulation of L-type and P/Q-type Ca2+ channel activity is an important mechanism to control Ca2+ entry into excitable cells. Here we addressed the question whether the activity of E-type Ca2+ channels can also be controlled by Ca2+. Switching from Ba2+ to Ca2+ as charge carrier increased within 50 s, the density of currents observed in HEK-293 cells expressing a human Cav2.3d subunit and slowed down the inactivation kinetics. Furthermore, with Ca2+ as permeant ion, recovery from inactivation was accelerated, compared to the recovery process recorded under conditions where the accumulation of [Ca2+]i was prevented. In a Ba2+ containing bath solution the Ca2+-dependent changes of E-type channel activity could be induced by dialysing the cells with 1 micro m free [Ca2+]i suggesting that an elevation of [Ca2+]i is responsible for these effects. Deleting 19 amino acids in the intracellular II-III linker (exon 19) as part of an arginine-rich region, severely impairs the Ca2+ responsiveness of the expressed channels. Interestingly, deletion of an adjacent homologue arginine-rich region activates channel activity but now independently from [Ca2+]i. As a positive feedback-regulation of channel activity this novel activation mechanism might determine specific biological functions of E-type Ca2+ channels.

International audienceThe transverse tubule system in mammalian striated muscle is highly organized and contributes to optimal and homogeneous contraction. Diverse pathologies such as heart failure and atrial fibrillation include disorganization of t-tubules and contractile dysfunction. Few tools are available for the quantification of the organization of the t-tubule system. We developed a plugin for the ImageJ/Fiji image analysis platform developed by the National Institutes of Health. This plugin (TTorg) analyzes raw confocal microscopy images. Analysis options include the whole image, specific regions of the image (cropping), and z-axis analysis of the same image. Batch analysis of a series of images with identical criteria is also one of the options. There is no need to either reorientate any specimen to the horizontal or to do a thresholding of the image to perform analysis. TTorg includes a synthetic "myocyte-like" image generator to test the plugin's efficiency in the user's own experimental conditions. This plugin was validated on synthetic images for different simulated cell characteristics and acquisition parameters. TTorg was able to detect significant differences between the organization of the t-tubule systems in experimental data of mouse ventricular myocytes isolated from wild-type and dystrophin-deficient mice. TTorg is freely distributed, and its source code is available. It provides a reliable, easy-to-use, automatic, and unbiased measurement of t-tubule organization in a wide variety of experimental conditions

Pasqualin C, Gannier F, Yu A, Malécot CO, Bredeloux P, Maupoil V. SarcOptiM for ImageJ: high-frequency online sarcomere length computing on stimulated cardiomyocytes. Am J Physiol Cell Physiol 311: C277-C283, 2016. First published June 22, 2016; doi:10.1152/ajpcell.00094.2016.-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 Ͼ10 kHz. In conclusion, SarcOptiM is a free and validated user-friendly tool for studying cardiomyocyte contraction in all species, including human. cardiomyocyte contractility; sarcomere dynamic; video analysis; ImageJ plug-in; fast Fourier transform ISOLATED CARDIOMYOCYTE (CM) contractions can be recorded and measured with two main methods: cell shortening and sarcomere shortening. The latter is probably the most common and reliable technique used to characterize isolated CM contractile performance, because it does not depend on cell shape and size (4). Sarcomere shortening technique has applications in different research fields, including cardiovascular physiology, pathophysiology such as heart failure, pharmacology, and toxicology (2, 3, 6 -8, 11).Under transmission light microscopy, striated muscle cell sarcomeres show a transverse pattern due to the alternation of light (isotropic; I) and dark (anisotropic; A) bands, corresponding to the very regular organization of thin filaments of actin associated with regulatory proteins, such as tropomyosin and troponin (I band) and thick filaments of myosin (A band). These bands have a profile that can be assimilated to a sinusoidal curve. The frequency of this sinusoid, which represents the distance between the dark bands of the myosin filaments and, therefore, the sarcomere length (SL), can be extracted from Fourier spectrum analysis of the CM image.

Na channels confer a TTX-sensitive basal Na(+) permeability to rat PV cardiac muscle cells and contribute to the slope of slow depolarization between bursts of CAA. Na channel blockers act mostly via reduction of the Na window current. Ranolazine also has an anti-α1 adrenergic effect, which contributed to its antiarrhythmic effect.

There is growing evidence that E-type voltage dependent Ca(2+) channels (Ca(v)2.3) are involved in triggering and controlling pivotal cellular processes like neurosecretion and long-term potentiation. The mechanism underlying a novel Ca(2+) dependent stimulation of E-type Ca(2+) channels was investigated in the context of the recent finding that influx of Ca(2+) through other voltage dependent Ca(2+) channels is necessary and sufficient to directly activate protein kinase C (PKC). With Ba(2+) as charge carrier through Ca(v)2.3 channel alpha(1) subunits expressed in HEK-293 cells, activation of PKC by low concentrations of phorbol ester augmented peak I(Ba) by approximately 60%. In addition, the non-inactivating fraction of I(Ba) was increased by more than three-fold and recovery from short-term inactivation was accelerated. The effect of phorbol ester on I(Ba) was inhibited by application of the specific PKC inhibitor bisindolylmaleimide I. With Ca(2+) as charge carrier, application of phorbol ester did not change the activity of Ca(v)2.3 currents but they were modified by the PKC inhibitor bisindolylmaleimide I. These results suggest that with Ca(2+) as charge carrier the incoming Ca(2+) can activate PKC, thereby augmenting Ca(2+) influx into the cytosol. No modulation of Ca(v)2.3 channels by PKC was observed when an arginine rich region in the II-III loop of Ca(v)2.3 was eliminated. Receptor independent stimulation of PKC and its interaction with Ca(v)2.3 channels therefore represents an important positive feedback mechanism to decode electrical signals into a variety of cellular functions.

The effects of synthetic poneratoxin (PoTX), a new toxin isolated from the venom of the ant Paraponera clavata, were studied under current- and voltage-clamp conditions in frog skeletal muscle fibres. PoTX induces a concentration-dependent (10(-9) M-5 x 10(-6) M) prolongation of the action potentials and, at saturating concentration, a slow repetitive activity developing at negative potentials. PoTX specifically acts on voltage-dependent Na channels by decreasing the peak Na current (INa) and by simultaneously inducing a slow INa which starts to activate at -85 mV and inactivates very slowly. Both the fast and the slow components of INa are suppressed by tetrodotoxin and reverse at the same potential corresponding to the equilibrium potential for Na ions. The fast component of INa has voltage dependence, activation and steady-state inactivation almost similar to those of the control INa. The voltage dependence of the slow Na conductance is 40 mV more negative than that of the fast one. The results suggest that PoTX affects all the Na channels and that the fast and the slow INa components originate from a possible PoTX-induced interconversion between a fast and a slow operating mode of the Na channels.