The intrinsic cellular mechanisms by which length regulates myocardial contraction, the basis of the Frank–Starling relation, are uncertain. The aim of this work was to test the hypothesis that passive force, possibly via titin, participates in the modulation of Ca2+sensitivity of cardiac contractile proteins induced by stretch. Titin degradation by a mild trypsin digestion modulated passive force induced by increasing from 1.9 to 2.3 μ m sarcomere length in skinned rat cardiac cells. Force–pCa curves were established at these two sarcomere lengths after various durations of trypsin application that induced different passive force levels. They allowed us to evaluate myofilament Ca2+sensitivity by the pCa of half-maximal activation (pCa50). In control conditions, stretching cells from 1.9 to 2.3 μ m induced a leftward shift of pCa50(ΔpCa50) of 0.39±0.03 pCa units (mean±SEM, n=8 cells), reflecting an increase in Ca2+sensitivity of the contractile machinery. Passive force measured every 2 min decreased exponentially after the beginning of the trypsin application (t1/2@12 min). The first 30% decrease of passive force did not affect the stretch-induced variation in Ca2+sensitivity. Then, with further decrease in passive force, ΔpCa50decreased. At the lowest passive force investigated 20% of initial passive force, ΔpCa50decreased by approximately 55%. These effects were not accompanied by a significant modification of either maximal activated force at pCa 4.5 solution or pCa50at 1.9 μ m sarcomere length. This indicates that there was no major functional alteration of the contractile machinery during the protocol as also suggested by contractile and regulatory protein electrophoresis on 2.5–12% gradient and 15% SDS–PAGE gels. Thus, besides modulation induced by the reduced lattice spacing during enhanced heart refilling, Ca2+sensitivity of the cardiac contractile machinery may be controlled at least partially by internal passive load, which is known to be largely attributable to titin.

We studied active and passive properties of intact isolated guinea-pig ventricular myocytes in auxotonic conditions. Cells were attached using carbon fibres. The passive properties of the myocytes, in the presence of the stretch-activated channel blocker streptomycin sulphate, could be separated into two groups: stiff cells (stiffness slope = 2.88 +/- 0.93 nN/micron3, n = 63 cells) and compliant cells (stiffness slope = 0.91 +/- 0.35 nN/micron3, n = 52 cells). The study and the localization of the different kind of cells indicated that endocardium is mainly constituted of stiff cells (80%) while the epicardium contained more compliant cells (60%). When a longitudinal strain was applied to compliant cells, an increase in resting tension, diastolic sarcomere length and active tension were observed. On the other hand, in stiff cells, it induced an increase in resting tension and active tension with little change of diastolic sarcomere length. In both kinds of cells, strain had no effect on Ca2+ transient amplitude and shape. Plotting active tension v diastolic sarcomere length also clearly showed two separated populations of cells, corresponding to stiff and compliant cells. The results of the two groups of cells when plotting active tension v resting tension could not be distinguished. We conclude that resting tension is an important factor in the modulation of active tension by stretch in addition to interfilament lattice spacing or sarcomere length.

Objectives: The aim of the study was to investigate the mechanisms responsible for provoking and maintaining a large, ig 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+](0) and application of the Ca2+-channel blocker verapamil (1 mu M). The phenomenon was not sensitive to disruption of sarcoplasmic reticulum function by ryanodine (1 mu M) nor to the Na+ channel blocker TTX (30 mu M). 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 Ca2+-window current maintain the increased levels of resting [Ca2+](i).

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.

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.

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 μM and a complete inhibition at 10 μM 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.

A mixture of crude collagenase and non-specific proteases has been used to isolate guinea pig ventricular heart cells. Measurements of collagenase activity with Wünsch's 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.

We developed an easy to use and non-invasive method to study sarcomere motion of enzymatically isolated myocytes which can be simultaneously combined with auxotonic force detection, thus being very useful when studying the contractile performance of cardiac cells. This method basically consists in analyzing the periodicity of the cell striation pattern using the Cooley-Tukey fast Fourier transform (FFT) algorithm on a video image of the cell during the course of the experiment. A longitudinal fraction of the cell image is recorded with a CCD TV camera, digitized, then transiently stored on a computer and used to calculate the spectrum corresponding to the distribution of the sarcomere lengths (SL). The method gives a real-time measurement of the most probable value of sarcomere length in one isolated cell with a temporal resolution of 20 ms. When used on a cell attached between two carbon fibers, the auxotonic force developed by the cell upon electrical stimulation can be simultaneously measured together with the SL in various conditions of stretch. Preliminary results have been presented in abstract form (Gannier et al., vol 24, pp. S47, 1992).

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.

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.