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J Physiol Volume 560, Number 2, 403-411, October 15, 2004 DOI: 10.1113/jphysiol.2004.062604
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Contraction augments L-type Ca2+ currents in adherent guinea-pig cardiomyocytes

Uwe Rueckschloss1 and Gerrit Isenberg1

1 Department of Physiology, Martin Luther University, 06097 Halle, Germany


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
As integrins are thought to function as mechanoreceptors, we studied whether they could mediate mechanical modulation of the L-type Ca2+ channel current (ICa) in guinea-pig cardiac ventricular myocytes (CVMs). CVMs were voltage clamped with 280 ms pulses from –45 to 0 mV at 0.5 Hz (1.8 mM [Ca2+]o, 22°C). Five minutes after whole-cell access (designated as 0 min) peak ICa was determined from a current–voltage (I–V) curve. Additional recordings were made after 5, 10 and 15 min. At control, ICa was not stable, but ran down during these periods. This run-down of ICa was attenuated by soluble fibronectin (FN) and was changed to an enhancement of ICa when CVMs were attached to FN-coated coverslips. Soluble peptide containing the integrin binding sequence of FN, Arg-Gly-Asp (RGD motif), did not modulate ICa; however, ICa increased in stimulated CVMs attached to RGD peptide-coated coverslips. The effect was not specific to integrins, because attachment to poly-D-lysine-coated coverslips also augmented ICa in stimulated CVMs. Augmentation of ICa by immobilized FN required rhythmical contraction of attached CVMs, because it was attenuated without electrical stimulation and after cell dialysis with the calcium chelator BAPTA. Furthermore, contraction-induced augmentation of ICa in FN-attached CVMs was sensitive to inhibition of protein kinase C (PKC; by Ro-31–8220), inhibition of tyrosine kinase activity (herbimycin A) and cytoskeletal depolymerization (cytochalasin D or colchicine). We attribute augmentation of ICa to the activation of signalling cascades by shear forces that are generated when CVMs contract against attachment; in vivo similar signals may occur when CVMs contract against attachment of integrins to the extracellular matrix.

(Received 5 July 2004; accepted after revision 3 August 2005; first published online 5 August 2004)
Corresponding author U. Rueckschloss: Department of Physiology, Martin Luther University, 06097 Halle, Germany. Email: uwe.rueckschloss{at}medizin.uni-halle.de


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Pressure overload-induced cardiac hypertrophy is known to be at least partially mediated by changes in the Ca2+ homeostasis of cardiac myocytes. Persistent elevation of cytosolic Ca2+ concentration was shown to stimulate the activity of Ca2+/calmodulin-dependent kinase and the protein phosphatase calcineurin (Braun & Schulman, 1995; Klee et al. 1998). Both of these contribute to the induction of hypertrophic gene expression by activation of transcription factors (Molkentin et al. 1998; Passier et al. 2000).

Several mechanisms can induce a persistent elevation of cytosolic Ca2+ concentration. There is evidence for a crucial role of Ca2+ influx through L-type calcium channels in this process, as it was shown that activation of calcineurin in angiotensinII- and phenylephrine-induced cardiac hypertrophy was attenuated by the application of calcium channel blockers (Kato et al. 2000; Huang et al. 2002). Stretch of cardiomyocytes was shown to modulate calcium transients (Petroff et al. 2001) and mechanical regulation of cardiac calcium handling proteins, including L-type calcium channels, is implied by studies using agents that interfere with the integrity of the cytoskeleton (Galli & DeFelice, 1994; Kerfant et al. 2001; Rueckschloss & Isenberg, 2001). Therefore, it is reasonable to speculate that there may be a contribution of L-type calcium channel currents (ICa) to an altered Ca2+ homeostasis in pressure overload-induced cardiac hypertrophy.

Mechanical stress is the primary stimulus in pressure overload-induced cardiac hypertrophy (Cooper et al. 1985). So it is important to know which molecular structure senses and transduces mechanical stress and how the resulting signal is linked to L-type calcium channels. Integrins are thought to act as mechanoreceptors (Hynes, 1992). These membrane receptors are heterodimers composed of different {alpha}- and ß-subunits. In the heart a variety of {alpha}-subunits, that seem to associate exclusively with splice variants of the ß1 isoform, are expressed (for review see Ross & Borg, 2001). Integrins bind different components of the extracellular matrix thus contributing to the anchorage of the cell within the tissue. Intracellularly, these receptors couple to the actin-based cytoskeleton via linker proteins. Therefore, integrins would be able to sense and transduce external mechanical forces onto intracellular structures. In addition, ligation of extracellular matrix proteins to integrins induces signalling cascades independently of mechanical forces. It has been shown that stimulation of {alpha}5ß1 integrins with FN immobilized on microbeads augmented L-type calcium channel activity in vascular smooth muscle cells (Wu et al. 1998b). The augmentation of ICa required activation of focal adhesion kinase (FAK) and was mediated by src kinase-dependent phosphorylation of Tyr2122 in the C-terminus of the {alpha}1-subunit of the Ca2+ channel (Wu et al. 2001). However, this mechanism is unlikely to occur in cardiac myocytes, because there are substantial structural differences between smooth muscle and cardiac isoforms of the L-type calcium channel {alpha}1-subunit and because the critical Tyr2122 does not exist in the cardiac isoform.

In the present study, we show that the cardiac ICa is augmented when the cells are attached to coverslips coated with either FN or a synthetic peptide that contains Arg-Gly-Asp (RGD motif), the integrin binding sequence of fibronectin. The increase in ICa requires contraction of CVMs induced by continuous electrical stimulation. This effect is not mediated by the induction of integrin-specific biochemical signalling cascades, because non-specific adherence of CVMs to immobilized poly-D-lysine via electrostatic interactions similarly augments ICa in the presence of continuous electrical stimulation. We conclude that cardiac ICa is stimulated by mechanical forces that are induced when the CVMs contract against attachment. These forces are sensed by integrins and putatively transduced via the actin cytoskeleton.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell isolation and solutions

Adult guinea-pigs (~300 g) were killed by cervical dislocation in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85–23, revised 1985) and with the local ethics committee. Ventricular myocytes were isolated by a standard collagenase dissociation technique. During the experiment, the cells were continuously superfused with an extracellular solution composed of (mM): NaCl 150, CsCl 5.4, CaCl2 1.8, MgCl2 1.2, glucose 20 and Hepes 5 (pH 7.4). Whole-cell patch-clamp recordings were performed with pipettes (resistance, 2–3 M{Omega}) filled with (mM): CsCl 140, NaCl 5, MgCl2 0.5, EGTA 0.005 and Hepes 10 (pH 7.4). Chemicals were purchased from Sigma unless otherwise stated.

Coating of coverslips

Fibronectin (FN), fibronectin-like engineered protein polymer-plus (FN-like EPP) and poly-D-lysine were obtained from Sigma. Solutions of FN (0.1 mg ml–1) and FN-like EPP (0.05 mg ml–1) were allowed to completely dry onto the coverslips. Poly-D-lysine (0.5 mg ml–1) was removed after 1 h, coverslips were washed with deionized water and were dried. In all cases, 100 µl cm–2 was used. After insertion of the coated coverslip into the recording chamber, it was washed for 5 min with extracellular solution before introducing the cells. The cells were allowed to adhere to the coverslip for at least 30 min before recordings were started. Adherent cells were identified by lifting the patch electrode at the end of the recordings. Non-adherent cells could be lifted with the patch electrode, whereas adherent cells remained attached to the coverslip.

Measurement of whole-cell Ca2+ currents

Starting from a holding potential of –45 mV, 280 ms pulses to 0 mV were applied at 0.5 Hz. Currents were recorded with a RK300 amplifier (Biologic, Echirolle, France) connected to a personal computer via a CED–1401 interface (CED, Cambridge, UK). The peak current through L-type Ca2+ channels (ICa) was estimated as difference negative peak current minus late current at the end of the 280 ms pulse (see Isenberg & Klöckner, 1982). During the period of the experiment (20 min) access and seal resistance remained constant.

Statistics

Values are given as means ± S.E.M. Significance was determined by Student's unpaired t test and was assumed at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Spontaneous run-down of ICa

The measurements of ICa in isolated guinea-pig ventricular myocytes were started 5 min after whole-cell access. At this time point, a reference I–V curve was recorded, and peak ICa was determined and defined as 100%. Under control conditions, there was a run-down of ICa as indicated by the reduction of peak ICa over time. ICa was reduced to 90 ± 3%, 82 ± 4% and 71 ± 5% (n = 14) after 5, 10 and 15 min, respectively. This spontaneous run-down is in accordance with the literature (Kameyama et al. 1997).

Modulation of ICa by integrin stimulation with fibronectin

Modulation of ICa was analysed in response to soluble as well as to immobilized FN (Fig. 1). Soluble FN was applied immediately after the recording of the reference I–V curve. Infusion of Tyrode solution supplemented with 80 nM FN attenuated the spontaneous run-down of ICa to 92 ± 3%, 94 ± 4% and 95 ± 4% after 5, 10 and 15 min, respectively (n = 6; P < 0.05 versus control at 15 min; Fig. 2A). Normalized to the time-matched control, this represents a significant increase of ICa to 134 ± 5% at 15 min.



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  		Figure 1.  Representative original current traces and I–V curves of ICa from FN-treated CVMs
ICa was elicited by 280-ms pulses from –45 mV to 10 mV. Each plot contains two superimposed current traces that were recorded at 0 min (blue traces, maximum inward current labelled with a) and 15 min (red traces, maximum inward current labelled with b). Under control conditions (no interventions, continuous electrical stimulation by 280-ms pulses from –45 mV to 0 mV at 0.5 Hz), a decline of ICa over time was evident. This spontaneous run-down of ICa was reduced by application of soluble FN (80 nM). In CVMs that were immobilized on FN-coated coverslips, there was no reduction of ICa after 15 min (immobilized FN). Without continuous electrical stimulation, attachment of CVMs to FN-coated coverslips did not compensate for the spontaneous run-down of ICa. The I–V curves of ICa were recorded at 0 min (open circles) and at 15 min (red-filled circles). The reduction in peak ICa that was evident under control conditions was diminished by application of soluble FN. In CVMs attached to FN, peak ICa was unchanged only in the presence of continuous electrical stimulation.

 


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  		Figure 2.  Time-dependent changes in peak ICa by FN
I–V curves were recorded and peak ICa was determined at 0, 5, 10 and 15 min. A, the time-dependent decline of peak ICa in control CVMs was significantly reduced by application of soluble FN (80 nM) only after 15 min. B, immobilization of CVMs on FN-coated coverslips significantly augmented ICa at every time point. C, attachment of CVMs to FN-coated coverslips did not prevent time-dependent reduction of peak ICa without continuous electrical stimulation. *P < 0.05.

 
To immobilize CVMs on FN, coverslips of the recording chamber were coated with the protein. During the continuous stimulation with clamp pulses (0.5 Hz), ICa in adherent CVMs did not run down but increased to 106 ± 4%, 106 ± 6% and 104 ± 6% after 5, 10 and 15 min, respectively (n = 9; P < 0.05 versus control at all time points; Fig. 2B). Normalized to the time-matched controls, ICa was augmented to 147 ± 9% after 15 min. The values of ICa stimulation by soluble and immobilized FN are not statistically different at that time point.

Cell dialysis with ATP-free solutions could have contributed to the spontaneous run-down of ICa in ruptured patch experiments (Hao et al. 1999). Therefore, we analysed the stimulation of ICa by immobilization of CVMs on FN-coated coverslips in the presence of ATP. Inclusion of 4 mM Na2ATP (plus 4.5 mM MgCl2) into the electrode solution reduced spontaneous run-down of ICa. In controls, ICa fell to 100 ± 5%, 98 ± 6% and 94 ± 7% after 5, 10 and 15 min, respectively (n = 8). Stimulation of ICa in CVMs attached to FN was further augmented in the presence of ATP to 117 ± 5%, 122 ± 7% and 119 ± 9% after 5, 10 and 15 min, respectively (n = 8; P < 0.05 versus control in the presence of ATP at all time points).

Modulation of ICa by integrin stimulation with RGD-containing peptide

To analyse whether the RGD motif of fibronectin was sufficient to stimulate ICa, a synthetic peptide that contains multiple copies of the RGD motif of FN was used (FN-like EPP). In contrast to FN, application of 100 nM soluble FN-like EPP did not significantly modulate ICa. Peak ICa fell to 93 ± 5%, 89 ± 6% and 87 ± 5% within 5, 10 and 15 min, respectively (n = 8; n.s. versus control; Fig. 3A).



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  		Figure 3.  Time-dependent changes in peak ICa by FN-like EPP
A, compared to control CVMs, application of soluble RGD-containing synthetic peptide (FN-like EPP, 100 nM) did not significantly modulate peak ICa. B, in CVMs that were immobilized on FN-like EPP-coated coverslips ICa was significantly augmented at every time point. *P < 0.05.

 
Conversely, the effect of the immobilized synthetic peptide was similar to immobilzed FN. In adherent CVMs, ICa was augmented to 113 ± 7%, 110 ± 10% and 102 ± 11% after 5, 10 and 15 min, respectively (n = 6; P < 0.05 versus control at all time points; Fig. 3B). Thus, ICa increased to 143 ± 15% of the time-matched control after 15 min.

Stimulation of ICa by non-specific attachment of cardiomyocytes to immobilized poly-D-lysine

We wanted to adress the question of whether the stimulation of ICa by attachment of CVMs to immobilized integrin ligands is unique to this class of membrane receptors. Therefore, ICa was analysed in CVMs that attached to the coverslip by electrostatic interactions with immobilized poly-D-lysine, that is without induction of integrin-specific signalling cascades (Fig. 4). Also in these experiments, ICa was found to be strongly stimulated. In attached CVMs, ICa increased to 116 ± 7%, 123 ± 9% and 121 ± 11% within 5, 10 and 15 min, respectively (n = 8; P < 0.05 versus control at all time points; Fig. 5A). This increase corresponds to 169 ± 15% of the time-matched control after 15 min.



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  		Figure 4.  Representative original current traces and I–V curves of ICa from CVMs immobilized on poly-D-lysine
The two superimposed current traces of ICa on each plot were recorded at 0 min (blue traces, maximum inward current labelled with a) and 15 min (red traces, maximum inward current labelled with b). The spontaneous run-down of ICa under control conditions was attenuated in CVMs that were immobilized on poly-D-lysine-coated coverslips. Without continuous electrical stimulation, attachment of CVMs to poly-D-lysine-coated coverslips did not compensate for the spontaneous run-down of ICa. The I–V curves of ICa were recorded at 0 min (open circles) and at 15 min (red-filled circles). The reduction in peak ICa that was evident under control conditions was attenuated by attachment of CVMs to poly-D-lysine only in the presence of continuous electrical stimulation.

 


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  		Figure 5.  Time-dependent changes in peak ICa by immobilization of CVMs on poly-D-lysine
A, in comparision to control CVMs, peak ICa was significantly augmented in CVMs that were immobilized on poly-D-lysine-coated coverslips at every time point. B, the augmentation of peak ICa by immobilization of CVMs on poly-D-lysine is attenuated without continuous electrical stimulation. *P < 0.05.

 
Augmentation of ICa depends on contraction of CVMs

Binding of integrins to their ligands can induce biochemical signalling cascades that are independent from mechanical stimulation. If such signalling cascades were responsible for the stimulation of ICa, one would expect an increase of ICa density in CVMs that adhere to immobilized integrin ligands for extended periods of time without contraction of the cells. Therefore, at the beginning of the pulsing protocol we analysed ICa density in controls and CVMs that had been attached to coated coverslips for at least 30 min. There was no difference in ICa density between controls (9.4 ± 1.1 A F–1; n = 11), CVMs attached to FN (9.4 ± 0.9 A F–1; n = 10) or CVMs attached to poly-D-lysine (10.0 ± 0.8 A F–1; n = 15).

In all experiments, CVMs were pulsed from –45 mV to 0 mV at 0.5 Hz during the 5-min periods between the recordings of the I–V curves. That is, CVMs rhythmically contracted. In order to analyse whether these contractions are essential for the augmentation of ICa in attached CVMs, experiments were performed where the CVMs were not stimulated in the periods between the recordings of the I–V curves. Spontaneous run-down of ICa was unchanged by these experimental conditions. In controls, ICa fell to 95 ± 3%, 86 ± 4% and 71 ± 4% within 5, 10 and 15 min, respectively (n = 10). In contrast, stimulation of ICa in CVMs immobilized on FN (95 ± 6%, 92 ± 6% and 83 ± 7%, respectively, n = 6; n.s. versus control; Fig. 2C) as well as on poly-D-lysine (92 ± 8%, 85 ± 7% and 72 ± 7%, respectively, n = 5; n.s. versus control; Fig. 5B) was attenuated by this non-continuous treatment.

In order to block contraction in the presence of continuous electrical stimulation, control CVMs and attached CVMs had been dialysed with an electrode solution containing 5 mM BAPTA. This treatment not only attenuated spontaneous run-down but slightly increased ICa in control CVMs (110 ± 2%, 108 ± 4% and 106 ± 5% at 5, 10 and 15 min, respectively, n = 8) In the presence of BAPTA, attachment of CVMs to FN did not further augment ICa above control levels (109 ± 4%, 112 ± 7% and 112 ± 8% at 5, 10 and 15 min, respectively, n = 8, n.s. versus control in the presence of BAPTA).

Effect of cytochalasin D and colchicine on contraction-induced augmentation of ICa in FN-attached CVMs

We wanted to evaluate the role of the cytoskeleton in the contraction-induced augmentation of ICa in FN-attached CVMs. Therefore, ICa was analysed in non-attached and attached CVMs in the presence of compounds that interfere with the integrity of the cytoskeleton.

The F-actin disrupter cytochalasin D (10 µM) was applied via the bath solution for up to 1 h. In the presence of cytochalasin D, augmentation of ICa was attenuated in FN-attached CVMs. That is, ICa was not different in non-attached control cells (89 ± 6%, 74 ± 4% and 66 ± 9% at 5, 10 and 15 min, respectively, n = 3) and CVMs attached to FN (82 ± 9%, 79 ± 15% and 57 ± 7% at 5, 10 and 15 min, respectively, n = 3, n.s. versus control in the presence of cytochalasin D; Fig. 6A). Unfortunately, application of cytochalasin D reduced contraction of CVMs. In the presence of cytochalasin D, contraction was almost completely abolished 5–10 min after whole-cell access.



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  		Figure 6.  Time-dependent changes in peak ICa by cytochalasin D and colchicine
A, application of the F-actin disrupter cytochalasin D (10 µM) attenuated the augmentation of ICa in CVMs immobilized on FN-coated coverslips. B, run-down of ICa in control CVMs was compensated for in the presence of the microtubule depolymerizing agent colchicine (10 µM). Of more importance, attachment of CVMs to FN-coated coverslips did not further augment ICa in the presence of colchicine.

 
Incubation of control CVMs with the microtubule depolymerizing agent colchicine (10 µM) for at least 2 h compensated the run-down of ICa (105 ± 4%, 101 ± 5% and 97 ± 6% at 5, 10 and 15 min, respectively, n = 4). Of more importance, adherence of CVMs to FN-coated coverslips in the presence of colchicine did not superimpose on these changes (99 ± 8%, 96 ± 6% and 92 ± 5% at 5, 10 and 15 min, respectively, n = 4, n.s. versus control in the presence of colchicine; Fig. 6B).

Augmentation of ICa depends on PKC and tyrosine kinase activity

We have analysed the effect of PKC and tyrosine kinase inhibition on the contraction-induced augmentation of ICa in CVMs adherent to FN, because both signalling molecules are known to be sensitive to mechanical modulation.

In non-adherent control CVMs, intracellular application of the PKC inhibitor Ro-31–8220 (100 nM; Calbiochem) via the patch electrode compensated the run-down of ICa (104 ± 6%, 100 ± 7% and 96 ± 8% at 5, 10 and 15 min, respectively, n = 9). Adherence of CVMs to FN did not further augment ICa above these control levels (104 ± 5%, 106 ± 9% and 101 ± 9% at 5, 10 and 15 min, respectively, n = 9, n.s. versus control in the presence of Ro-31–8220; Fig. 7A).



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  		Figure 7.  Time-dependent changes in peak ICa by Ro-31–8220 and herbimycin A
In the presence of the PKC inhibitor Ro-31–8220 (100 nM; A) or the tyrosine kinase inhibitor herbimycin A (1 µM; B), run-down of ICa was compensated for in non-attached control CVMs. Attachment of CVMs to immobilized FN did not add to the effect of these inhibitor-induced changes. That is, ICa was not different in non-attached and attached CVMs.

 
Extracellular application of the tyrosine kinase inhibitor herbimycin A (1 µM; Calbiochem) also compensated run-down of ICa in non-adherent control CVMs (99 ± 4%, 104 ± 4% and 102 ± 3% at 5, 10 and 15 min, respectively, n = 7). Adherence of CVMs to FN did not superimpose on the herbimycin A-induced augmentation of ICa (98 ± 5%, 94 ± 4% and 89 ± 8% at 5, 10 and 15 min, respectively, n = 5, n.s. versus control in the presence of herbimycin A; Fig. 7B).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the present study we analysed the modulation of L-type Ca2+ currents by soluble and immobilized integrin ligands in CVMs. Using whole-cell recordings at room temperature, we found ICa to be augmented by soluble FN but not by a soluble synthetic peptide containing the RGD motif of FN (FN-like EPP). Further, ICa was augmented in CVMs immobilized on FN- as well as FN-like EPP-coated coverslips. Non-specific attachment of CVMs via electrostatic interactions of the sarcolemma with poly-D-lysine-coated coverslips also increased ICa. The augmentation of ICa critically depended on the rhythmical contractions of the attached cells and the integrity of the cytoskeleton, and was sensitive to inhibition of PKC and tyrosine kinase activity. We conclude that augmentation of ICa is due to mechanical interaction of the contracting cell with immobilized extracellular matrix proteins, a new aspect of integrin signalling in attached CVMs.

In vascular smooth muscle cells, ICa was shown to be augmented by a soluble peptide containing the LDV (Leu-Asp-Val) motif of an alternately spliced FN variant via {alpha}4ß1 integrin activation (Waitkus-Edwards et al. 2002). Immobilized FN that binds to {alpha}5ß1 integrins via the RGD motif was also shown to increase ICa in vascular smooth muscle cells (Wu et al. 1998b). Although different integrins were stimulated, in both cases tyrosine kinase activation was involved. The present modest augmentation of cardiac ICa by soluble FN could also be due to the LDV-dependent {alpha}4ß1 integrin stimulation. This is supported by the finding that the soluble synthetic peptide (FN-like EPP) that contained the RGD motif of FN in multiple copies, but not the LDV motif had no significant effect on cardiac ICa.

Augmentation of ICa by FN immobilized on the coverslip was both faster and stronger than by soluble FN. Furthermore, the synthetic peptide that failed to augment ICa in its soluble form, strongly augmented ICa in CVMs attached to FN-like EPP-coated coverslips. The augmentation of ICa by immobilized FN is not a reversal of the spontaneous run-down caused by ATP depletion because the effect of attachment of CVMs to FN superimposed on the ATP-mediated augmentation of ICa.

As the critical Tyr2122 for src kinase-dependent augmentation of ICa in vascular smooth muscle cells is missing in the cardiac {alpha}1 isoform, a different signalling pathway has to be involved.

Several lines of evidence suggest that the augmentation of ICa in immobilized contracting CVMs represents a new mechanism distinct from the well-established biochemical integrin signalling. Firstly, despite the fact that CVMs had been allowed to attach to the immobilized FN for at least 30 min, there was no difference in ICa density at the beginning of the pulsing protocol between non-attached controls and attached CVMs. Secondly, ICa was also augmented without induction of integrin-specific signalling cascades by attachment of CVMs to immobilized poly-D-lysine, an effect that can not be induced by application of soluble poly-D-lysine (Shepherd et al. 1990). Therefore, we conclude that augmentation of ICa in attached CVMs is independent of signalling cascades that are triggered when specific ligands bind to integrins. It is interesting that in CVMs either attached to FN or attached to poly-D-lysine, ICa was only augmented when the cells rhythmically contracted. Thus, neither sole binding of immobilized ligands to integrins nor putative effects of poly-D-lysine on sarcolemma surface charges alone could be responsible for the observed augmentation of ICa in immobilized CVMs. It is unlikely that step depolarization itself is responsible for the stimulation of ICa in attached CVMs, because the same stimulation protocol was applied to non-attached controls and attached CVMs. Furthermore, ICa was not different in non-attached controls and attached CVMs that had been dialysed with BAPTA, that is in the presence of repetitive electrical stimulation but without contraction. Only attachment in conjunction with the mechanical stimulus of contraction-dependent shortening was sufficient to inrease ICa. Therefore, we suggest that the asymmetrical attachment changes mechanical forces due to contraction of CVMs into shear forces, because shortening would be restricted to the non-attached side of the CVMs. Similar shear forces are suggested to occur in vivo, because the myocardium is organized in laminae or sheets of cardiomyocytes separated by cleavage planes that contain the collagen network (LeGrice et al. 1995a) and relative sliding of these myocardial laminae was demonstrated (LeGrice et al. 1995b) during passive filling at increasing pressures in rat hearts (Spotnitz et al. 1974).

How could these mechanical forces modulate ICa? One putative mechanism is the modulation of ICa by the mechanical forces itself. Recently, a protein called AHNAK was shown to possess binding sites for the regulatory ß-subunit of L-type calcium channels as well as for actin filaments (Hohaus et al. 2002). Although highly speculative, AHNAK could serve as a linker between the channel and the actin cytoskeleton, thus permitting the transfer of mechanical forces from sites of attachment directly onto the channel protein via the actin cytoskeleton.

Alternatively, mechanical forces induced by contraction of adherent CVMs could have modulated enzymatic activities that in turn stimulate ICa. PKC and tyrosine kinase activity have been shown to contribute to mechanotransduction in cardiomyocytes (Sadoshima et al. 1996; Seko et al. 1999). Therefore, we analysed ICa in contracting FN-attached CVMs in the presence of Ro-31–8220 or herbimycin A. Both interventions compensated for the spontaneous run-down of ICa in non-attached CVMs. This is unexpected, because so far neither PKC nor tyrosine kinase activity have been linked to this phenomenon. It is more important that in the presence of PKC and tyrosine kinase inhibition, attachment of CVMs to FN did not augment ICa above the values of inhibitor-treated control CVMs. That is, PKC and tyrosine kinase inhibition attenuated the response of ICa to contraction in attached CVMs. These results support an indirect mechanism of ICa stimulation via contraction-induced modulation of signalling cascades.

The transmission of forces from sites of attachment to signalling molecules could be mediated by the cytoskeleton. Therefore, we have analysed ICa in CVMs treated with agents that interfer with the integrity of the cytoskeleton. In the presence of the F-actin disrupter cytochalasin D, the augmentation of ICa in FN-attached CVMs was attenuated. Unfortunately, cytochalasin D also reduced contractions of CVMs in our experiments, an effect that is in accordance with the literature (Wu et al. 1998a). Therefore, we conclude that the contraction-induced stimulation of ICa in FN-attached CVMs is sensitive to cytochalasin D, but it is impossible to ascribe the attenuated response of ICa to the cytochalasin D-induced reduction in contraction (reduced stimulus) or to the cytochalasin D-induced breakdown of the cytoskeleton (reduced transmission of stimulus).

Application of the microtubule depolymerizing agent colchicine did not interfere with contraction of CVMs. In the presence of colchicine, run-down of ICa was compensated for in non-attached CVMs. With regard to the ruptured patch configuration used in our study, this observation is in accordance with the literature (Calaghan et al. 2001). Of more importance, in the presence of colchicine ICa was similar in non-attached control CVMs and CVMs adherent to FN-coated coverslips. That is, depolymerization of microtubules attenuated augmentation of ICa due to contraction of FN-attached CVMs. These results support a crucial role of the cytoskeleton for the mechanically induced augmentation of ICa in attached CVMs, putatively because it transmits mechanical forces from sites of attachment to signalling molecules.

Collectively, these data indicate an augmentation of cardiac L-type Ca2+ channel activity by mechanical forces that result from the interaction of the contracting attached ventricular myocyte with its extracellular surrounding. The present results indicate that the underlying mechanism of ICa augmentation does not depend on integrin-specific signalling cascades, but requires PKC and tyrosine kinase activity. We suggest that integrins could function as mechanical linkers to the extracellular matrix proteins that transduce the mechanical forces to the actin cytoskeleton. The augmentation of ICa by increased mechanical interaction of the myocyte with its surrounding extracellular matrix may contribute to an altered Ca2+ homeostasis in pressure overload-induced cardiac hypertrophy.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Braun AP & Schulman H (1995). The multifunctional calcium/calmodulin-dependent protein kinase: from form to function. Annu Rev Physiol 57, 417–445.[CrossRef][Medline]

Calaghan SC, Le Guennec J-Y & White E (2001). Modulation of Ca2+ signaling by microtubule disruption in rat ventricular myocytes and its dependence on the ruptured patch-clamp configuration. Circ Res 88, E32–E37.[Medline]

Cooper G IV, Kent RL, Uboh CE, Thompson EW & Marino TA (1985). Hemodynamic versus adrenergic control of cat right ventricular hypertrophy. J Clin Invest 75, 1403–1414.[Medline]

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   Acknowledgements
 
This work was supported by the Deutsche Forschungs-gemeinschaft Sonderforschungbereich/Transregio 02.




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