Isolated Cardiomyocytes: Mechanosensitivity of Action Potential, Membrane Current and Ion Concentration

Isenberg G, Kondratev D, Dyachenko V, et al.

Publication Details

Isolated cardiomyocytes can be mechanically stretched by a variety of methods that are reviewed. The cell's electrophysiological response to a homogeneous axial stretch is relatively small, however, it is large during a local stretch that applies an additional shear stress component. If not voltage-clamped, cardiomyocytes respond local stretch with membrane depolarization, prolongation of the action potential duration and eventually with extra systoles. Under voltage clamp, local stretch induces an inward current (ISAC) through non-selective channels and it deactivates the inwardly rectifying K+ current (IK1), both changes contributing to the arrhythmogenicity. ISAC is reviewed in terms of ion selectivity and mechanosensitivity. Putative activation mechanisms require an intact cytoskeleton, details, however, are speculative at present. Stretch increases the concentrations of Na+ and Ca2+ in the cytosol and in the cell organelles, partly because of the Na+- and Ca2+ influx carried by ISAC. The modulation of long-lasting stretch on development of hypertrophy and cardiac failure are not included in this review. Instead, the consequences of stretch-induced ion accumulation are discussed in regard to the slow component of the force staircase, and the consequence of stretch-modulated currents in terms of stretch-induced arrhythmias.


The first experiments demonstrating the significant influence of mechanical factors on cardiac excitations and contractions were obtained in vivo. Eventually, coronary catheterization induced extra action potentials that could trigger arrhythmias of the re-entry type [53]. Later, the effect of mechanical stretch was studied in isolated hearts by inflating a balloon in one of its chambers while the resulting changes in the action potential were recorded with surface electrodes [48]. Essentially, these studies indicated that mechanical stretch of the ventricular wall reduced the action potential duration by activating K+ channels of the ATP-sensitive type [69].

This brief review reports on recent work investigating the effects of mechanical deformation on the ionic currents in isolated cardiomyocytes, and it describes the stretch-induced changes in resting and action potential on the results of these voltage clamp experiments. The isolated cardiomyocytes have the advantage that one can combine analysis of membrane currents with analysis of fluorescence signals suitable to quantify possible stretch-induced changes in the cytosolic Ca2+ concentration [Ca2+]c. Thus, the single cell is a useful model for the study of cellular mechanisms of action, and to combine it with information on the length-dependent mechanical properties of cardiomyocytes [76]. However, it is less suitable to study those stretch-effects that depend on interaction of the cardiomyocytes with their neighbours and with the proteins of the extracellular matrix; those effects are not included in this review. Also not included are long-term events such as development of hypertrophy and failure.

Methods to stretch the isolated cardiomyocyte

The problem to stretch an isolated adult cardiomyocyte is not trivial. There is not one single method of stretch but at least 8 various ways, all of them are technically demanding and with a relatively low success rate. Fabiato [13] isolated pairs of myocytes in tandem, he mechanically fixed a force transducer to one of the cells that was damaged thereby whilst the other was surviving. The method was given up because of its low success rate. Attachment of a force transducer to a single cardiomyocyte by glue did not succeed because all these glues damaged the cell membrane (see comments in [81]).

  1. Local stretch. Single myocytes have been stretched with a pair of patch pipettes (PP). The first PP squeezes part of cell to the coverslip, the other PP with a wide opening lifts and stretches the myocyte [61, 73, 82].
  2. Method 1) was modified by replacing the second PP with a fire-polished glass stylus that adhered to the cell surface via electrostatic interactions [7]. This method of local stretch displaced the glass stylus together with part of the cell surface and underlying sarcomeres (Fig. 1A). It does not stretch the bottom of the cell that was attached to the glass bottom of the chamber where attachment was facilitated coating it with poly-L-lysine. Thus, the method of local stretch applies beside a component of axial stretch a component of shear stress [31].
  3. Homogeneous axial stretch of the sarcomeres was achieved by sucking both cell ends into the openings of 2 glass pipettes whose distance could be varied by piezo devices [81]. A third manipulator was used to impale a PP for electrophysiology (Fig. 1B).
  4. Several groups succeeded in attaching a thin carbon fibre to the cell end, presumably by electrostatic interaction (Fig. 1C). When the fibre was moved together with the cell end, an almost homogenous lengthening of the sarcomeres could be achieved [46, 77, 78].
  5. Paramagnetic microbeads coated with antibodies against integrins were loaded to the cell surface. After attachment and binding, the bead was moved by means of a magnetic force, thereby locally deforming the surfacemembrane (Fig. 1D, [4]).
  6. Isolated myocytes were embedded with an argarose gel in thin wall polyethylene tubing's, stretching the tubing stretched the gel together with the cells [56, 66]. This method was used to apply fluorescence indicators, it does not allow the insert a patch electrode and is not suitable for electrophysiology.
  7. Ventricular myocytes were seeded and attached to Bioflex® membranes covered with collagen or fibronectin. The commercial device applies a vacuum to stretch the membrane rhythmically, in both in x and y to the same extent, and the attached cells are stretched together with the membrane [29, 42]. Since the flexible membranes are not transparent but milky, cell or electrode are barely visible, and neither electrophysiology nor fluometry has been combined with this type of stretch.
  8. Cell swelling by pressure inflating the cell or via exposure to hypoosmotic extracellular media can provide a three-dimensional stretch of the cell. The method has the problem that changes in volume are not easily related to changes in tension. The literature agrees that it is difficult to distinguish between the effects of the stretch component and other possible effects of swelling, i.e. the changes in electrophysiology can't be compared with those due to homogenous or local stretch.
  9. Single channel analysis. A cell attached or cell free patch of membrane can be stretched by applying positive or negative pressure to the open end of the patch electrode. In some publications, video microscopy could analyze the size and shape of the membrane patches in the recording patch pipettes [65]. They were therefore able to calculate the membrane tension from the transmembraneous pressure and the curvature of the patch (Lapace Law).

Figure 1. Methods to stretch isolated ventricular myocytes.

Figure 1

Methods to stretch isolated ventricular myocytes. A) Local axial stretch. The right part of the cell is pressed by the patch pipette (PP) versus the coverslip and hence fixed. The stylus (S) adheres to the cell surface electrostatically. When the distance (more...)

Although all methods impose a mechanical force to the cell, the type of this force differs. Thus, one should not be surprised if the final readout, i.e. the change in cellular electrophysiology, is not identical. In this review we concentrate on a comparison between results obtained by local and by homogenous axial stretch. As mentioned above, the methods of homogenous axial stretch (3 and 4) apply a force that is oriented parallel to the cell axis, thereby intending to lengthen the sarcomeres as they may do during cardiac filling. The methods of local stretch (methods 1, 2 and 5) impose in addition to axial stretch a shear force. They induce spatial inhomogeneities within the cell and its cytoskeleton, a situation that may be caused by cardiac impact during an accidence (ice hockey) or during catheterization. In this review, we will try to explain discrepancy between the stretch effects on action potentials and membrane currents with the different force components applied to the myocytes.

Stretch changes the cardiac resting and action potential

Fig. 2 illustrates the basic effects of stretch on membrane resting and action potentials. Panel A shows recordings from a human heart where monophasic action potentials (APs) were recorded with surface electrodes put on the right endocardium [48]. The upper trace C labels the control situation. The patient was undergoing pulmonary valvuloplasty, when surgery acutely obstructed the right ventricular outflow tract (S), action potentials had a longer duration, resembled afterdepolarizations (AD, arrow) from which extra systoles could arise (ES, arrow).

Figure 2. Stretch effects on membrane resting and action potential (AP).

Figure 2

Stretch effects on membrane resting and action potential (AP). A: Monophasic AP recordings from right endocardium in a patient undergoing pulmonary valvuloplasty. Control (C) versus response to acute obstruction of right ventricular outflow tract (S), (more...)

Panel B shows an AP from a guinea pig ventricular myocyte before (C) and during an axial stretch (S) that lengthened the sarcomere spacing from 1.85 μm to 2.11 μm (method 3 [5]). The changes are relatively small; the resting potential is by 2 mV more positive and the duration of the AP is 7% longer. Panel C shows recordings from a guinea-pig ventricular myocyte that was locally stretched by 4 μm. That is, the distance between the glass stylus and the patch pipette was increased by 4 μm from 35 to 39 μm. This increased the sarcomere length in the line between PP and S increased from 1.84 up to 1.98 μm, outside this line the sarcomere length was almost constant (Fig. 1B). The small mechanical distortion induced the resting membrane to depolarized by 8 mV. The repolarization of the AP was speeded up during phase 1 and retarded during phase 2. Due to this "cross over", the APD (0 mV) was shortened by local stretch, and the APD (90 mV) was 20% prolonged. When the same cell was locally stretched by 8 μm, a pacemaker-like depolarization (3) appeared that reached the threshold and started an extra systole (ES). The following stimulus found the cell in the relative refractory period, as the consequence, the next systole (action potential NS) was reduced in amplitude and duration. Fig. 2D could be the single cell correlate of a situation generating ventricular arrhythmias in vivo. Occasionally, the effects of local stretch could be investigated in ventricular myocytes from human cardiac explants. Basically, the effects resembled those described for the guinea pig ventricular myocyte (Fig. 2E). Surprisingly, the sensitivity of the sick human cells to local stretch was very high. A small local stretch of 2 μm already induced a large depolarization, a shortening of phase (1) and a prolongation of phase 2 repolarization of the action potential (see below [36]).

In summary, the extent of the stretch-induced changes in the ventricular resting and action potential depend on the methods of stretch. Typical for homogenous axial stretch is a retardation of the plateau phase without obvious effects on the rate of repolarization during phase 1 and 2 that are induced with a shear force component. We suggest that homogenous and local axial stretch are different types of mechanical stimuli.

Stretch modulates the net membrane current

The changes of the action potential result from the mechanosensitivity of several ionic current components as they can be measured by the voltage clamp technique. Fig. 3A compares the net membrane currents before (blue) with those during a 10 μm local stretch (red). Between the pulses the membrane potential was held at -45 mV, and a holding current of +0.2 nA was measured. The first step to +10 mV (70 ms, see line at the bottom of Fig. 3A) induced a time-dependent current wave. The initial negative current surge of -0.2 nA reflects the contribution of an L-type ICa. Inactivation of ICa and activation of the potassium current IKo increases the net late current (IL) at the end of the pulse to +0.6 nA. Repolarization to the holding potential brings the current back to 0.2 nA. The final step to -80 mV goes along with a negative tail current (IT) that changes with time; this is presumably the contribution of the Na+,Ca2+-exchanger. Within 40 ms the current becomes close to zero (+0.04 nA) corresponding to the resting potential of -80 mV. During the stretch (Fig. 3A, red trace), the holding current at -45 mV became negative (IH = -0.34 nA). The pulse to 10 mV induced a peak of net inward current that was close to the zero line. With time, the current increased to IL =0.76 nA at the end of pulse. At -45 mV, the current was again -0.3 nA. The second step to -80 mV induced a large inward current IT = -0.80 nA, equivalent to the stretch-induced membrane depolarization. The effect of stretch is further visualized with the difference currents (Fig. 3B) where the current at control has been subtracted from the current during local stretch. The difference current is almost time-independent, despite a little outward hump at the beginning of the pulse to +10 mV which reflects a small reduction of ICa (see below). The stretch-induced difference currents are -0.85 nA at -80 mV, -0.56 nA at -45 mV and +0.11 nA at +10 mV.

Figure 3. Net membrane currents modulated by local axial stretch.

Figure 3

Net membrane currents modulated by local axial stretch. A: Currents in response to 120 ms clamp steps that depolarize from -45 mV (holding potential) for 70 ms to either +10 mV or to -80 mV. Back trace at bottom indicates the pulse protocol. Black tracing (more...)

Since local stretch seemed to add on the control currents a current that was almost time-independent, ramp-like clamp commands were used to study the voltage-dependence of the net membrane currents, i.e. the potential was set to +60 mV and than repolarized to -100 mV at a rate of 100 mV/s. The resulting iv curve is a plot of the late current versus the respective voltage. At control (Fig. 3C, blue), the curve intersects the voltage-axis at a "zero current potential" of -88 mV that corresponds to the resting potential of this cell. The curve is N-shaped, showing a hump of outward current between -60 mV and -50 mV. Stretch by 5 μm (red curve) shifted the iv curve at negative potentials downwardly. That is, the new zero current potential of -70 mV reflects the stretch-induced membrane depolarization. Up to -20 mV, the amount of outward current is reduced, a correlate for the stretch-induced retardation in repolarization phase 2. At positive potentials, the currents are increased by stretch. This effect, together with the attenuation of ICa, is the correlate for the faster repolarization phase 1. If we compare the stretch induced net currents with the reports in the literature, they differ in dependence on the method, similar as described above for the stretch-induced changes in the action potential. There are only a few manuscripts reporting stretch induced changes in the whole cell current, i.e. for homogenous axial stretch [5, 11, 26, 81] and for local axial stretch [7, 36, 37, 44, 61, 81, 82].

The separation of the net current (Fig. 3) into its ionic components is difficult, since we expect most ion channels to be mechanosensitive [59]. It is unlikely that the stretch sensitive currents presented with Fig. 3 result from a selective stretch modulation of only one type of ion channel. For didactical reasons, we distinguish a "direct" mechanosensitivity of the ion current from an "indirect" one. We use prefix "indirect" to indicate that the respective current is not changed primarily by a change in channel open probability but because of a changed driving force, stretch may have induced ion accumulation.

In terms of the current amplitude, currents evoked by local stretch (shear stress) are larger than those evoked by axial stretch. A recent study [5] reported that stretching the sarcomeres from 1.85 μm to 2.11 μm induced negative currents in the order of -0.1 or less, in correlation with the minor depolarization and modest prolongation of the AP (Fig. 2B). Axial stretch of 3 μm between the 2 glass pipettes induced time-dependent inward currents of -1pA/pF at -100 mV, a current density that may be translated into an amplitude of -0.1 nA per cell. At -100 mV, non-axial stretch was reported to induce negative currents of -0.4 nA in the small atrial myocytes [82]. In guinea-pig ventricular cells, the current was in the order of -0.6 nA [61]. A non-axial stretch by the stylus induced approx. -0.2 nA at -70 mV [7].

Regarding a possible time dependent gating, we did not see a time dependent decline in the current amplitude as expected from the single channel recordings ([59] for review) or from smooth muscle cells [74]. Instead, the inward current was at a stable amplitude for periods as long as 10 min. It has been described that the glass stylus should attach to the cell surface for several minutes before local stretch can induce currents, and that then the current started always with a spiky (shorter than 5 ms) inward current before a stable plateau was reached [7]. Although our own method resembles to the one of this paper, we never have seen such a phenomenon.

ISAC, the stretch-activated current through non-selective cation channels

According to the literature, there exists a type of ion channels that is gated only by mechanical stimuli and not by changes in the membrane potential. These stretch activated channels (SACs) were first described in skeletal muscle fibres [57, 63] and oocytes [14, 45, 80] when a negative pressure (suction) was applied to the patch pipette during the formation of the Giga-Seal. We refer to currents through these channels as stretch activated non-selective cation currents and follow the proposed abbreviation (ISAC).

To separate ISAC from the net membrane current, the other current components should be blocked. In ventricular myocytes, block of superimposed K+ currents can be achieved by substituting the K+ ions by Cs+ ions in both the extra- and the intracellular space. For the intracellular substitution, the cell is dialyzed with an electrode solution where Cs+ ions are the dominant cations. In the extracellular space one can substitute the 5 mM K+ by 5 mM Cs+, thereby blocking the inwardly rectifying K+ channel [30].

Voltage dependence of ISAC

To understand the voltage-dependence of the stretch activated current, we plotted the currents flowing at the end of the 140 ms pulses (late currents IL) as iv curves versus the potential (Fig. 4A). Before the mechanical stimulus (filled circles), the resulting iv curve was flat small because Cs+ had blocked the K+ current components, the slope at negative potentials indicated a high input resistance of 500 MOhm. A 10 μm local stretch displaced the iv curve to the open circles (input resistance 62.5 MOhm). During stretch, the iv curve was almost linear and intersected the voltage axis at -5 mV. Panel B of Fig. 4 shows the difference current, i.e. the current induced by stretch. In this definition, ISAC showed an almost linear voltage-dependence, i.e. its conductance GSAC was constant (no dependency on membrane potential). The data of Fig. 4B could be fitted by linear regression where the conductance was 16.5 nS and the reversal potential was Erev = -0.5 mV (r=0.996).

Figure 4. Voltage dependence of the stretch induced current ISAC through non-selective cation channels.

Figure 4

Voltage dependence of the stretch induced current ISAC through non-selective cation channels. Currents at the end of 70 ms pulses were plotted versus the potential of the clamp pulse. Superimposed K+ currents were blocked by replacing K+ by Cs+ ions in (more...)

Linear iv curves with reversal potentials between 0 and -15 mV were reported for both axial stretch [5, 81] and for local stretch [36, 37, 82]. Since at potentials negative to -10 mV ISAC is inward, its activation can depolarize the diastolic membrane potential, and it can retard the repolarization phase 2, from the plateau back to the resting potential. At positive potentials ISAC is outward. This stretch activated outward current can shorten the initial repolarization phase 1. Thus, the properties of the mechanically activated current ISAC can explain the stretch induced modification of the resting and action potentials in non-clamped cardiomyocytes.

ISAC is a non-selective cation current

ISAC was characterized in terms of the ion selectivity. Substitution of chloride by aspartate or by glutamate did not change amplitude or reversal potential suggesting that movement of Cl--ions does not contribute to ISAC, or that the channel is a cation selective channel [7, 36, 81]. To quantify the relative permeability of the stretch activated channel for different cations, the 150 mM extracellular Na+ ions were replaced by the same concentration of an other cation species, resulting in a change of both amplitude and reversal potential of ISAC. From the shifts we estimated that the SACs are best permeable for Cs+ ions [36, 82]. Usually, the reversal potential of ISAC is approx. -10 mV, this would indicate that K+ efflux and Na+ influx pass through the channel with the same permeability. For mouse ventricular myocytes superfused with a Ca2+ free medium, the permeability ratio was PCs: PK :PNa :PLi :PTMA :PNMG = 1.3 :1.1 :1.0 :0.8 :0.1 :0.03 [37]. That is, even the large organic cations tetramethyl ammonium (TMA+) or N-methyl glucosamine (NMG+) could carry a small inward current when local stretch activated SACs. The numbers can't be easily compared with others because of different temperatures (22 °C in [81, 82]) and extracellular Ca2+ concentrations in the extracellular superfusate (0 mM in [37], 0.2 mM in [81] and 1.8 mM in [82]) and the patch electrode (low or high concentrations of EGTA or BAPTA, see below). In rat atrial myocytes [82] the permeability ratio was PCs :PNa :PLi =1.05 : 1 : 0.98 for the stretch activated current and PCs :PNa :PLi= 1.49 : 1.0 : 0.79 for the non-selective background current.

In guinea pig ventricular cells, stretch induced a negative ISAC also when the 150 mM Na+ ions were replaced by 90 mM Ca2+. At -100 mV, there was a Ca2+ carried inward current that reversed polarity at +15 mV as one would expect for Ca2+ ions passing inwardly at the same ease as K+-ions flowing outwardly. However, the iv curve was no longer linear with Ca2+ as the charge carrier, negative to -20 mV it bended to become nearly independent of membrane potential [36]. In murine ventricular myocytes isotonic substitution of Na+ by Ca2+ induced outward current components that hampered the quantification of ISAC and its Ca2+ permeability. In summary, we conclude that SACs are Ca2+ permeable, albeit with low permeability (compare [73], for review see [21, 59]).

When Ca2+ was removed from an extracellular Cs+ solution during continuous stretch, the amplitude of ISAC increased by a factor of 2.5 as if Ca2+ ions would hinder the permeation of the Cs+ ions [37]. An even stronger hindrance was achieved by addition of the 3-valent cations La3+ or Gd3+, both blocked ISAC. The concentration for this block was low, 8 μM were saturating provided one waited for 10 min. This contrasts with the 100 μM concentrations used in other reports; we suggest that these high concentrations are the result from the fact that the on-rate of the block increases with the concentration (low of mass action). The Gd3+ block of ISAC was not completely reversible, i.e. ISAC did not recover within 30 min, as can be expected from the high affinity of Gd3+. The block of SACs by divalent and trivalent cations has been described in the literature [79]. It has been interpreted as a fast flickering block, i.e. the permeating cation interacts with the inner side of the channel protein; the single channel current is interrupted for the time of interaction (binding). If the channel is blocked for several femto seconds, this block appears as a reduced current amplitude if the recording system has a time resolution in the order of milliseconds.

In rat atrial myocytes, two types of non-selective channels have been described, SACS and non-selective background channels. Gadolinium inhibited the background channels in a concentration dependent manner with an IC50 value of 46 μM. In presence of 100 μM Gd3+, stretch still induced ISAC and diastolic depolarization [82]. The result can't be explained in the context of the other publications, at the moment. The question whether or not SACs are open without the application of exogenous stretch, would have consequences for the cellular Na+ homoeostasis (see below), for ventricular myocytes a clear answer is missing. For the experiments applying axial stretch to guinea-pig ventricular myocytes, the answer would be no channel openings without sarcomere lengthening [5], and Gd3+ was reported not to hyperpolarize the resting membrane [25]. This conflicts with the Gd3+ induced hyperpolarization reported by those groups that applied local stretch [36, 37, 82]. Also Fig. 4A indicates that Gd3+ shifted the iv-curve in the continuous presence not back to the control before stretch but to less inward current (triangles more positive than filled circles in Fig. 4A). At present, one can not rule out that part of the difference is due to the mechanical effects of attaching the glass stylus, even if the stylus had not been used for stretch.

Unfortunately, Gd3+ is not a selective blocker of SACs, it reduces also the potassium currents IK1 and IKo [25]. Hence, 30 μM streptomycin has been introduced as tool to separate ISAC from the stretch induced net currents [5, 17]. ISAC strongly resembled when these currents were defined as difference current by 8 μm Gd3+ or by 30 μM streptomycin [44]. In both definitions, ISAC had a voltage-independent conductance and a reversal potential of approx. -10 mV. Some groups have used the drug amiloride to block SACs in oocytes [20], also the selectivity to SACs is low, it binds to a variety of other channels. For purification of the channel protein a highly specific toxin would be desirable. A peptide isolated from the spider Grammtula spalata (GTx) has been identified and shown to be useful [67]. Unfortunately, the effort to synthesize this protein has not yet been successful. For a recent review on the pharmacology of mechanogated channels see [23].

Single channel analysis

The first evidence for the existence of SACs came from single channel recordings performed on oocytes [45, 80], myoblasts [19], smooth muscle cells [73] and embryonic chicken heart cells [57, 63]. In most experiments, the single channel current was activated by negative pressure (suction) applied to the open end of the electrode forming the cell attached patch (Fig. 5). The open probability could be enhanced equally well by suction or by pressure. There was no obvious time delay between suction and appearance of channel activity. The SACs in oocytes were cation-selective with PK >PCs >PNa >PLi >PCa [80]. Tetramethylammonium was impermeable. In smooth muscle cells, single channel conductance followed the rank PK >PNa >PCs >PBa >PCa [24]. The single channel conductance was between 20 pS and 40 pS, depending on the species of the permeable cation and on the temperature of the experiment.

Figure 5. Currents flowing through single SACs.

Figure 5

Currents flowing through single SACs. Left part, AA-AH: recordings from tissue-cultured chick ventricular myocytes, membrane hyperpolarized by 40 mV. Aa no suction, Ab 20 mm Hg suction. Ac, Ad amplitude histograms demonstrating the stretch activation (more...)

Results from single channel analysis in oocytes [21, 80] and embryonic chicken skeletal muscle fibres [19] described a time dependence of SACs. Upon step like changes in the negative pressure, SACs activate within several milliseconds [22] and than inactivate with a time constant in the order of 200 ms [52]. This inactivation could or could not lead to a sustained channel activity. Kinetic analysis indicated that there were three closed states and one open state. The open time was independent of both pressure and membrane potential, however, dependent on the ionic species [80]. The single channel kinetics should be reflected in the time course of whole cell currents, however, this link to the physiological significance of adaptation of single SACs has not yet been achieved [59].

There were only very few reports that reported successful single channel analysis in atrial myocytes [39, 82]. In ventricular myocytes the reports remain "episodic" [57] although several groups have spend long periods of unsuccessful experiments to find them. Nowadays one interprets that in ventricular myocytes SACs may be "hidden" in parts of the surface membrane where the patch pipette has only limited access, such as the transversal tubules [28] or the caveolae. Alternatively, it has been suggested that cytoskeletal structures that may be important for the mechanosensitivity, are destroyed when the membrane seals to the glass pipette [59].

The amplitude of ISAC and "mechanosensitivity"

Usually, the amplitude of the measured membrane current is normalized by the cell size, in the literature. For this, one measures the membrane capacity (proportional to the surface membrane) and divides the current by the given number of pF. In our own experiments, the membrane capacity varied between 76 and 135 pF, in dependence on species and age. Division of ISAC by 100 pF yields current densities between 5 and 20 pA/pF [36, 37], in dependence on membrane potential, extent of local stretch, and species (see below). These values are larger than those reported for end-to-end stretch (0.5–1.5 pA/pF) [5, 81]. We hesitate to apply the normalization process in case of the local stretch because this method modifies only part of the surface membrane. Essentially, this fraction is not known, it ranges between 5 and 20% of the outer cell surface. That is, the actual divisor should be smaller and the current density higher than given by the above numbers. An other estimate tries to find how many open channels (No) are necessary to generate a current of -1 nA at -100 mV, when the single channel conductance is 25 pS (see above). Using No = 1 nA/(25 pS*0.1 V) we obtain an estimate that 400 channels should open simultaneously.

To define the mechanosensitivity of whole cell ISAC, the amplitude of ISAC has been plotted versus the mechanical input. In case of axial stretch, the current at -100 mV increased linearly according to ISAC = -0.3*SL% (Fig. 6D, SL%: relative increase in the sarcomere length [81]). In case of local stretch [36], the relation was curvilinear. The slope of the curve defines the mechanosensitivity. Fig. 6C indicates that the mechanosensitivity was not constant but increased with the extent of local stretch. Fig. 6 also indicates that mechanosensitivity differs between ventricular myocytes isolated from hearts of different animals. Low mechanosensitivity was found for cardiomyocytes from mice and young rats (filled circles). Cardiomyocytes from young guinea pigs (empty circles) and rats in the age of 15 months (filled squares) had a higher mechanosensitivity. Cells from rats with spontaneous hypertension (SHR, 15 months, filled triangles) had a mechanosensitivity that was several times higher than the one of the age-matched controls (compare the third with the second trace in Fig. 6A). Also the human ventricular myocyte displayed a high mechanosensitivity (Fig. 6B, Fig. 6C open squares). The literature suggests that cardiac hypertrophy goes along with a proliferation of the subsarcolemmal cytoskeleton in the individual hypertrophied cardiomyocytes [72]. We speculate that these results may suggest that both cytochalasin D and colchicine soften cytoskeletal network; in the consequence the coupling between the mechanical stimulus and the activation of SACs would be less efficient.

Figure 6. Mechanosensitivity of ISAC measured at -45 mV holding potential (currents due to 140 ms depolarizations to 0 mV superimposed).

Figure 6

Mechanosensitivity of ISAC measured at -45 mV holding potential (currents due to 140 ms depolarizations to 0 mV superimposed). A,B: pen recordings, period and extent of local stretch indicated. A: Increase in mechanosensitivity in rat ventricular myocytes (more...)

How does mechanical deformation modulate channel activity?

Activation and inactivation of ion channels could be caused by mechanically induced changes in the surface tension in the lipid bilayer that would induce conformational changes in the channel protein. Such a mechanism has been made plausible for channels incorporated in the membranes of bacteria (MscL channel of E.coli, for details see the review [21]). For eukaryotes this explanation is unlikely because the membrane lipids are fluid, and there redistribution would rapidly abolish possible tension. In ventricular myocytes, stretch is suggested to unfold the 'slack' surface membrane primarily from subsarcolemmal invaginations near the z-line [47] and to induce incorporation of sub-membrane caveolae [40]. Hence, lengthening of the sarcomeres does not necessarily increase the stress in the lipid bilayer membrane. It is more likely that the structures of the extracellular matrix (ecm) and/or the cytoskeletal proteins bear and transmit the tension [58, 59]. In case of isolated ventricular myocytes the ecm is cleaved away, including the glycocalix [33], hence, the channel activation seems to be independent of ecm proteins. Activation of Cl--channels by pulling microbeads succeeded only when these beads were coated with antibodies binding to integrins. Further more, experiments studying the influence of mechanical stress on cellular hypertrophy plated the cells on matrix proteins interacting with integrins. Thus, mechanical stimuli may activate different targets via different signalling pathways.

When SACs were first described in embryonic chicken myotubes, the cells were treated with tubulin and actin reagents to test the involvement of the cytoskeleton. It was shown that colchicine depolymerising tubulin had no significant effect, but cytochalasins depolymerising F-actin increased the channel's stretch sensitivity [19]. The results suggested that neither tubulin nor f-actin would be necessary for mechanosensitive gating. It was postulated that the SACs were normally linked into a component of the membrane skeleton that runs parallel to the actin network, so that when actin was depolymerised by cytochalasin D, more stress could be transferred to the channel-linked component. The identity of that component has not yet been resolved [59]. Our lab tested colchicine and cytochalasin D on whole cell ISAC of murine ventricular myocytes [37]. Both toxins reduced steady state ISAC activated by stretch. Further, preincubation with one of the toxins prevented the mechanical activation of ISAC. The result that the basic effects of colchicine or cytochalasin D on ISAC did not differ, was somewhat unexpected. If depolymerization of tubulin and F-actin has very similar effects, mechanosensitivity is unlikely to be caused by a specific interaction between the SAC with one of these cytoskeletal elements [34]. That is, SAC opening should not be attributed to a direct interaction with tubulin or F-actin (protein-protein interaction).

At present, we speculate with the tensegrity model that both actin and tubulin are necessary for the mechanical tension within the cell. The tensegrity model [71] compares the surface membrane of a cell, e.g. of a fibroblast, with the tent supposing that tubulins were the stiff rods bearing the external mechanical load and the F-actin strands were the cables that are necessary to provide the tension. The intermediate filaments were supposed to be tension-free, until the cell would be deformed or would swell. The tensegrity model postulates that the exogenous mechanical energy would be distributed to all intracellular components including the nucleus. Whilst the model has been nicely elaborated for fibroblasts or endothelial cells, details applicable to cardiomyocytes are missing. That is, at present we do not know how much of the exogenous force is dissipated by the cytoskeleton and how much by the sarcomeric proteins (titin, proteins of the z-band etc.). If we apply the tensegrity model to cardiomyocytes, the above similarity of the effects of colchicines and cytochalasin would suggest that activation of SACs needs tension in the cell surface, or, that removal of this surface tension by breaking down one of the essential cytoskeletal constituents will hamper the spread out of energy from the stylus to distant structures. When the single channel activity was facilitated this would mean that local interactions within an isolated membrane patch would still function, and that cytochalasin has impaired the dissipation of mechanical energy from the patch to its neighbourhood.

The cellular elements involved in mechanical channel activation, i.e. mechanical deformation of the cell surface, stress propagation and modulation of protein conformation, may localize in different parts of the cell. In the example of endothelial cells, the streaming blood deforms the luminal surface, cellular cables transport this mechanical stimulus to the focal adhesions on the basolateral side of the cell, where integrins interact with the proteins of the extracellular matrix. The mechanical stimulus on these ligated integrins induces phosphorylation cascades that finally activate the NO-synthase that is localized in the caveolae of these cells. In cardiomyocytes, most have activated SACs with "naked tools" (fire polished glass, naked carbon fibres), i.e. channel activation seems to be independent of interaction between matrix proteins and integrins. An analogy of this signalling pathway has been suggested for rat ventricular myocytes: stretch can induce fluorescence signals indicative for the production of NO [56]. Using specific inhibitors, the likely signalling pathway is that axial stretch (sarcomere length increased from 1.81 to 1.99 μm) activates PI(3)kinase that phorphorylates Akt and eNOS in turn. Part of the stretch-induced signals would be caused by NO. NO is considered as a second messenger that can increase the open probability of the Ca2+ release channel RyR2 by nitrosylation [56]. Whether this NO dependent pathway would apply to activation SACs has to be clarified by future experiments.

Stretch-sensitive potassium currents

Cardiomyocytes bear a variety of potassium channels. For simplicity, one may distinguish along the voltage dependence inwardly from outwardly rectifying currents. The most important inward rectifier is the current IK1 (channel Kir2.1) that determines the repolarization phase 2 from the plateau and the stable resting potential of the ventricular myocytes. The conductance GK1 can be blocked by extracellular Cs+ ions [30]. Also the outwardly rectifying K+ current is composed of several components, here we summarise these Cs-insensitive K+ currents with IKo.

Mechanosensitivity of the inward rectifying current IK1

To study the possible mechanosensitivity of IK1 one has to superfuse the cells with "normal" extracellular solutions, e.g., K+ ions have to present in both patch electrode and bath. Under those conditions, IK1 generates the positive current hump at -60 mV to the N-shaped iv curve of the cell (Fig. 7, blue curve). Local axial stretch reduced this hump of outward current and reduced the slope of the iv curve, indicative for reduction of IK1. Separation of IK1 from other current components such as ISAC is possible because of their peculiar voltage dependence: The conductance GK1 deactivates with positive potentials (Fig. 7B, blue curve, scaled on the left ordinate), in contrast to GSAC that is voltage-independent.

Figure 7. Mechanosensitivity of K+ currents.

Figure 7

Mechanosensitivity of K+ currents. A: Iv curves of net membrane currents measured with ramp commands repolarizing the cell from +60 to -100 mV at a rate of 100 mV/s. Blue trace before, red trace during 10 μm stretch. Arrows mark the shift of resting potential (more...)

During control, the iv curve is determined by the current IK1 (Fig. 7C). During axial stretch, the current is shifted to the negative direction (Fig. 7A, red curve). In part, this effect is due to the induction of ISAC (Fig. 7D, blue curve). Stretch activation of ISAC, however, should have increased the slope conductance at negative potentials whilst stretch had reduced the conductance. Also, the reduced hump of the N-shaped curve can not be explained by activation of ISAC with a voltage-independent conductance GSAC. With both arguments, we modelled the stretch-induced changes in the membrane currents by superimposition of a mechanically deactivated IK1 on ISAC and could fit the results (Fig. 7D). The results indicate that local stretch reduces GK1 without changing its voltage-dependence. Such an effect could mean that the availability of the K1-channel is the parameter that is mechanosensitive. Unfortunately, GK1 is reduced also by low concentrations of Gd3+, hence, a pharmacological separation between the two mechanosensitive currents ISAC and IK1 has not yet been performed. The observed mechanosensitivity of IK1 contributes to the membrane depolarization of the diastolic potential and to the retardation of the repolarization phase 2. In line with the missing effect of axial stretch on this part of repolarization (Fig. 2B), the current recorded during axial stretch did not indicate a component due to reduced IK1. In the embryonic cell cultures from rat or chicken, stretching the cell did not affect IK1 [27, 61], however, in these non-adult cells IK1 amounts only to a fraction of its counterpart in adult mammalian cardiomyocytes.

Mechanosensitivity of the outwardly rectifying currents IKo

The literature on reconstituted K+-channels describes that mechanical deformation can activate a K+ current through TREK-1 channels [50, 55]. Single channel analysis has demonstrated the activation of this channel by positive pressure (and by arachidonic acid) in rat ventricular myocytes [38]. We speculate that the stretch-induced increase in outward current seen at positive potentials (olive curve between Fig. 7C, D) could be caused by a channel like this. In addition, mechanical deformation has been described to activate the ATP-depletion K+ current IK,ATP [69]. Also this K+ current could contribute to the increase in outward current at positive potentials, illustrated in Fig. 7A. In adult rat atrial myocytes, a stretch-activated single K+ channel (probably of the 2P domain K+ channel) could be analyzed in cell attached patches [54]. The mean conductance was 65 pS (-60 mV, 150 mM [K+]o). The open probability of this channel increased with the amplitude of suction, saturating at -50 mm Hg. The channel did not activate immediately but after a latency of 100 ms. Time to peak was 500 ms, then, the current inactivated to a plateau of 20% within 1 s.

Other mechanosensitive current components

In principle all membrane channels should respond to mechanical stimuli. One would expect that the mechanical energy modulates the energy barriers between the conformational states of a channel protein. Indeed, longitudinal stretch modifies the gating mechanism of Na+ channels [68]. Further, mechanical energy can modulate the conformation of proteins that are associated to cytoskeletal elements, such as F-actin, [34]. This could also mean that some stretch-sensitive kinases (Src, MAP) or phosphatases are activated that modulate the channel protein by phosphorylation/dephosphorylation in turn. Finally, stretch may alter the rate by which reactive oxygen species or NO are produced, and the channel gating may be modified by oxidation or nitrosylation (suggested for the stretch effects on Ca2+ release from the SR [56]). Here, only two further examples shall be presented.

The L-type Ca2+ current ICa generates the negative current wave seen during the initial 50 ms of the depolarizing clamp step (Fig. 3A). It generates the long lasting plateau of the action potential. Local mechanical stretch reduced ICa. The reduction was small within the initial seconds (Fig. 3A) and increased with the period of stretch [36]. This reduction was seen over the whole range of clamp potentials, i.e. the voltage dependence of ICa seems to remain unmodified (Fig. 8A). Whilst ISAC was insensitive to chelation of cytosolic Ca2+, the stretch-induced reduction of ICa disappeared after dialyzing the cell with 20 mM BAPTA from the patch pipette (Fig. 8B) [37]. The result corroborates with the report of homogenous axial stretch, applied to BAPTA-AM loaded guinea pig ventricular myocytes, that did not modify the amplitude of voltage dependence of peak ICa [5]. Hence, it is unlikely that the Ca2+ channel protein would directly interact with the cytoskeleton. Rather, we interpret this stretch-effect on ICa as a "secondary one" caused by stretch-induced increments in [Ca2+]c followed by Ca2+-mediated inactivation of the Ca2+ channel (Ca2+-Calmodulin interaction with the Ca2+ channel α subunit). That is, we postulate that the primary stretch effect increases [Ca2+]c via increase in Ca2+ influx through SACs or via Ca2+ release from the SR in the close neighbourhood of the L-type Ca2+ channel. ICa could be increased by cell swelling, either with hypotonic solutions or by inflating the cell with a positive pressure at the patch pipette [51].

Figure 8. Secondary mechanosensitivity of the L-type Ca2+ channel current ICa.

Figure 8

Secondary mechanosensitivity of the L-type Ca2+ channel current ICa. A: Iv curves of peak ICa before stretch (empty circles) and during a 10 μm stretch (filled circles), ICa is reduced by 40%. B: After dialyzed of 20 mM BAPTA from patch pipette (more...)

Stretch increases the cellular sodium concentration [Na+]c

Results from fluorescence indicators

Enhanced Na+ influx through SACs is expected to increase the cytosolic concentration [Na+]c. We have measured stretch-induced increments in [Na+]c by means of fluorescence indicators and by means of electronprobe microanalysis (EPMA). Fig. 9 shows pseudoratiometric images of [Na+]c, using Sodium Green® as the indicator. Before local axial stretch, the image was flat, i.e. the Na+ concentration within the cell was homogenous. A 5 μm stretch increased the fluorescence suggestive for increments in [Na+]c. These increments grew with the period of stretch. During a 3 min stretch, fluorescence was on average 2-fold higher than before stretch. Most importantly, stretch induced spatial heterogeneities in [Na+]c, i.e. there were patches with low and elevated [Na+]c close together. Fig. 9C shows these patches with more than doubled fluorescence as white spots. Unexpectedly, these white spots occurred not only close to the stylus or close to the patch pipette but also far away from the glass tools. Calibration of the fluorescence signal is problematic. Our own ratiometric SBFI measurements yield 11 mM for the isolated murine cardiomyocytes (16 mM in the literature [6]), and during 3 min stretch the local concentration could increase up to 30 mM (white-colored patches in Fig. 9C).

Figure 9. Stretch-induced increments in free cytoplasmic Na+ concentration [Na+]c analyzed by Sodium Green fluorescence in a camera imaging system.

Figure 9

Stretch-induced increments in free cytoplasmic Na+ concentration [Na+]c analyzed by Sodium Green fluorescence in a camera imaging system. Guinea pig ventricular myocyte stimulated at 2 Hz. A: Image before stretch, position of the glass stylus (S) and (more...)

Electronprobe microanalysis

Electronprobe microanalysis (EPMA) can quantify the elemental concentrations with the high lateral resolution of the electron microscope. EPMA measures the sum of free and bound sodium concentration, we abbreviate this concentration with σ[Na]. σ[Na] was nearly two-fold higher than [Na+]c estimated from fluorescence indicators. During control, σ[Na] was 23 ±1.8 mM in the peripheral and 17 ±0.8 mM in the central cytosol (see Fig. 11). In the 20 nm narrow subsarcolemmal space σ[Na] could be as high as 100 mM [64, 75]. A 3 min stretch of 5 μm increased σ[Na] in the peripheral cytosol to 48 ±5 mM and in the central cytosol to 29 ±2.4 mM (factors 2.1 and 1.7, respectively). When SACs were blocked by 30 μM streptomycin, stretch did no longer increase σ[Na] in the central cytosol, in the peripheral cytosol the stretch effect was strongly attenuated. The blocker of the Na+, H+-exchanger, Cariporide®, had no significant effect on the stretch effects on σ[Na] [43].

Figure 11. Analysis of stretch induced changes in the total concentration (σ) of sodium (σNa) and calcium (σCa) with electronprobe microanalysis (EPMA).

Figure 11

Analysis of stretch induced changes in the total concentration (σ) of sodium (σNa) and calcium (σCa) with electronprobe microanalysis (EPMA). A: longitudinal cryosection of murine ventricular myocytes frozen after stimulation without stretch (more...)

A 3 min stretch of 5 μm increased σ[Na] in all cell compartments. It increased σ[Na] in both nuclear matrix (factor 1.97) and nuclear envelope (factor 1.65). These changes were insensitive to the treatment of the cell with Cariporide® (factor 2.14 and 1.94, respectively). Stretch doubled σ[Na] in central mitochondria. σ[Na] of peripheral mitochondria was already high before stretch (21.6 ± 4.3 mM), this value was not further increased by stretch (21.2 ± 3.3 mM).

Currents linked to accumulation of [Na+]c

Since Na+ ions are the predominant charge carriers of ISAC, mechanical activation of ISAC is expected to increase the cytosolic sodium concentration [Na+]c (see below). The increase would become apparent only if the Na+, K+-ATPase would not completely compensate the extra Na+ ion inflow. The Na+, K+-ATPase generates a pump current IP because the efflux of 3Na+ ions is coupled to the infflux of 2K+ ions. The stretch induced pump current could be measured. After a 3 min stretch period (Cs/Cs conditions to reduce superimposed current components), the net current did not recover directly to its respective value before stretch but with an outward current above control that persisted for approx. 2 min. This current was sensitive to the removal of Cs+ from or to the addition of strophanthidin to the bath. We interpret that stretch induced IP belongs to the currents with secondary stretch sensitivity, because it is caused by the stretch induced Na+ accumulation.

Another process linked to [Na+]c is the Na+, Ca2+-exchange. NCX produces an inward current Ix when Ca2+ ions are extruded at the expense of Na+ influx (coupling 1Ca2+ to 3Na+ ions). The stretch induced increase in [Na+]c is expected to shift the reversal potential of Ix from -20 mV to more negative potentials. At the normal diastolic potential of e.g. -80 mV, stretch-induced accumulation of [Na+]c should go along with an Ix that is reduced in amplitude but lasting for a longer period of time (see the tail current IT in Fig. 3B). At the positive plateau potentials, the Ca2+ influx coupled to Na+ efflux should be more pronounced. With both mechanisms, accumulation of [Na+]c and shift of the reversal potential of Na+, Ca2+-exchange contribute to the increased Ca2+ loading that happens when ventricular myocytes are stretched (see below).

The H+, Na+-exchanger NHE removes cytosolic protons from the cytosol on the expense of Na+ influx. Stretch induced intracellular Na+ accumulation should attenuate the driving force and the rate of H+ efflux, similar as described for NCX above. Since the coupling is electroneutral, no direct change of net membrane current can be measured. However, one expects a possible intracellular acidification that could modulate other conductances in turn.

Stretch increases the cellular calcium concentration [Ca2+]c

Fluorescence indicators

Increments in [Ca2+]c were among the first effects reported for local mechanical stretch (embryonic chick heart cells: [63]). Without simultaneous analysis of membrane currents for possible involvement of ISAC, those experiments are relatively easy to perform, and stretch induced increments in [Ca2+]c have been analyzed by both luminescence [2, 8, 12, 60] and fluorescence indicators. In isolated cardiomyocytes, increments in [Ca2+]c due to axial stretch have been measured in guinea-pig ventricular myocytes [26, 78] and discussed in context with the stretch activation of SACs [16]. Fig. 10 compares signals measured by confocal microscopy. Every 2 ms, a new line was scanned along the axis of the cell. Comparison between A and B indicates that local stretch moved the upper cell edge upwardly. The contractions were induced by clamp steps depolarizing 40 ms from -45 to 0 mV. Contraction is seen as edge movement, before stretch the isotonic shortening peaks at 74 ms and falls with a half decay time of 66 ms. During stretch, contraction peaks already at 61 ms, and the half decay time is reduced to 40 ms. The linescans show the change in Fluo4-fluorescence in the pseudocolor code. The global Ca2+ transients during control peak within 28 ms to 900 nM and decay with a half times of 163 ms. During local stretch, the peak of 1000 nM is reached within 24 ms, and the decay is slightly faster (144 ms). More importantly, during local stretch sparks occurred during diastole (yellow arrows in Fig. 8B, average from 6 cells: NPo =12 ±2 before and 20 ±4 during a 10 μm local stretch). A similar result, augmentation of peak amplitude and of number of sparks, has been reported for myocytes axially stretched in the tube [56]. Combining the stretch experiments with fluorescence measurements of NO and protein phosphorylation let the authors conclude that stretch phorphorylates and activates eNOS via activation of PI3K and Akt, and NO would increase the open probability of ryanodine-receptor by nitrosylation.

Figure 10. Stretch-induced changes in the cytosolic Ca2+ concentration [Ca2+]c as analyzed pseudoratiometrically in the confocal microscope.

Figure 10

Stretch-induced changes in the cytosolic Ca2+ concentration [Ca2+]c as analyzed pseudoratiometrically in the confocal microscope. Mouse ventricular myocyte loaded by 5 μM Fluo-4AM, depolarized from -45 to 0 mV for 40 ms at 1 Hz. Upper panels: (more...)

Electronprobe microanalysis

Due to the huge number of cytosolic Ca2+ binding proteins the total Ca concentration σ[Ca] in the central cytosol is with a diastolic value of 0.4 ± 0.05 mM 4000-times larger than the free concentration. The ratio of σCa = 400 μM, to [Ca2+]c = 0.1 μm is the buffering power of the cytosol of the ventricular cell. In the peripheral cytosol σ[Ca] was 0.57 ± 0.09 mM (Fig. 11). A 3 min local 5 μm stretch increased σ[Ca] 2-fold to 0.84 ± 0.07 mM in the central and 1.5-fold to 0.84 ± 0.09 in the peripheral cytosol (both changes significant). When stretch was repeated after block of SACs with 30 μM streptomycin, the effect of stretch on σ[Ca] was prevented in the periphery, however only attenuated in the centre of the cell. The latter result may suggest that local mechanical deformation changes σ[Ca] not only via Ca2+ influx through streptomycin sensitive SACs (most pronounced in the cell periphery) but also through augmented Ca2+ release from the SR (see above).


Stretch increased σ[Ca] not only in the cytosol but also in the nuclear matrix from 0.18 ± 0.06 to 0.30 ± 0.05 mM (Fig. 11). It is unlikely that this increase is due to a Ca2+ release from the nuclear envelope because in this compartment σ[Ca] increased during local stretch from 0.93 ±0.15 mM to 1.53 ±0.26 mM (significant, factor 1.64). The stretch-induced Ca-increment in these two compartments was not changed when Na+- and Ca2+-influx through SACs was blocked with 30 μM streptomycin. Hence, stretch may augment the σ[Ca] in nuclear matrix and envelope by modulating Ca2+ release from the SR and/or inhibiting Ca2+ extrusion via NCX to the extracellular space. There might be a stretch effect on σ[Ca] in the mitochondria, however, these compartments have so low σ[Ca] that significance was not achieved because of the detection limit of EPMA for Ca.


Axial stretch and the passive mechanical properties of the cardiomyocyte

In vivo, the sarcomere length increases from peak systole (1.65 μm) to lack (1.85 μm) and to end of diastole (2.2 μm) by approx. 30%. The passive and active mechanical properties of single cardiomyocytes have been reviewed by Brady [9]. More recently, it has been shown that passive tension is generated largely by the extracellular matrix (collagen) and by the sarcomeric protein titin [18]. In comparison with the sarcomeric proteins, elements of the cytoskeleton, such as desmin intermediate filaments and microtubules, contribute only little to the passive tension [18]. Studies using the carbon fibre technique have revealed many similarities between the mechanical performance of single cell and multicellular preparations. For example, the time course of relaxation was load dependent, stretch prolonged the time course of relaxation in mammalian heart trabeculae and myocytes [62, 77].

The Starling mechanism

Stretch increases in the twitch force of contraction by a length-dependent change in muscle geometry (overlap and lattice spacing between thick and thin filaments) and by increase the number of formed crossbridges, amplified by a tension-induced increase in the sensitivity of troponin C (TnC) for Ca2+ [1, 15]. The expected stretch-induced increments in the [Ca2+]c transient or in the Ca2+ influx through L-type Ca2+ channels were not detected [26]. A possibility would be that stretch increases the Ca2+ binding to TnC during diastole (increased systolic shortening), that during diastole extra Ca2+ ions will dissociate from TnC and will be extruded via NCX [35]. Since the Ca2+ efflux goes along with a negative current Ix, the plateau of the action potential will be prolonged [10, 41].

The Anrep effect

The immediate stretch-induced increase in force is followed by a slow increase in twitch force over the next 10 –15 min [1]. The Anrep effect is present in single cells, in terms of contraction and [Ca2+]c transients [77].

Results on the contribution of [Na+]c were at controversy. When measured with the fluorescent indicator SBFI, [Na+]c did not increase in cells during the Anrep effect [26]. This result contrasts with other results that are listed in section 8. Also multicellular trabeculae show a stretch-induced increase in [Na+]c during the Anrep effect [70].

The importance of Na+ influx through SACs for the stretch-induced [Na+]c accumulation, however, has not yet been widely acknowledged. Instead, an autokrine -parakrine effect was suggested to increase [Na+]c [3]: Stretch should stimulate the release of angiotensin II from the cardiomyocytes, interaction with ATI receptors should stimulate the secretion of endothelin I in turn. ETI binding to its G-protein coupled receptor should stimulate the activity of NHE with the consequence of an increase in [Na+]c, and via NCX of [Ca2+]c. The results are essentially based on the effectiveness of a series of drugs that interact with the individual steps of this cascade [3]. When the effects of local stretch were re-studied in isolated ventricular myocytes, block of NHE with Cariporide® did not interfere with the stretch-induced accumulation of σ[Na], in contrast to streptomycin that blocked SACs and the increase in σ[Na] [43]. Also, the isolated cardiomyocyte is surrounded by a huge build of solution, and the distance to the neighboured cell is so large that parakrine effects are difficult to imagine. Thus, there might be different signalling pathways induced by local stretch of the isolated myocyte and the axial stretch of multicellular specimen.

Stretch-induced arrhythmias

There is no evidence that the rhythmic 30% lengthening of the sarcomeres from peak systole (1.65 μm) to end diastole (2.20 μm) induces arrhythmias. This is in accord with the single cell experiments that applied homogenous stretch. As reported in section 3, homogenous axial stretch increased the duration of the action potential only less than 20%. Since this small effect could be blocked not only by streptomycin but also by loading the cell with the Ca2+ chelator BAPTA, it was postulated that this stretch induced APD prolongation is due to electrogenic efflux of Ca2+ ions via NCX ([10] see above).

If the large homogenous stretch has so little consequences for electrophysiology in contrast with the big effects of local stretch, most of the channel modulation might be caused by the shear stress component of the local stretch. This conclusion is not in contradiction to the results from single channel analysis because these experiments do apply stretch with large inhomogeneities (for review [28, 59]). It is in line with the clinical observation that ventricular arrhythmias due to a chest wall trauma (commotio cordis) correlate with the peak of diastolic left ventricular pressure that can be increased by the impact to 350 mm Hg [49]. This fast and local event is supposed to locally stretch some myocyte. The present results suggest that these arrhythmias are caused not only by activation of K+ATP-channels [69] but by other mechanosensitive currents such as ISAC, IK1 and IKo. We have shown that local stretch changes the action potential in a characteristical fashion (Fig. 2C, D), it shortened the APD(0mV) and prolonged the APD(-80 mV). Further, local stretch depolarized the cell and could induce extra systoles. The single cell turned out as a model where activation of ISAC, activation of IKo and deactivation of IK1 were found to contribute to these changes.

The single cell studies on the mechanosensitivity of electrical activity have produced new findings, in particular the identification of stretch-activated and stretch-deactivated channels. The ability to record mechanical and electrical activity simultaneously with changes in the concentrations [Ca2+]c and [Na+]c is likely to be a powerful tool in the study of the mechanoelectric feedback. The challenge for the future is on one side to characterize better the force components of the mechanical stimuli and the intracellular signalling cascades activated by them. On the other side, forthcoming analysis has to relate the single cell/single channel findings to the observations made in the whole heart.


This work was supported by the Deutsche Forschungsgemeinschaft (Is 24/19-1).


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