TY - JOUR
AU - Isenberg,, Gerrit
AB - Abstract Objective: We describe mechanically induced non-selective cation currents in isolated rat atrial fibroblasts, which might play a role as a substrate for mechano-electrical feedback in the heart. Methods: Isolated fibroblasts were used for voltage-clamp analysis of ionic currents generating mechanically-induced potentials. Fibroblasts were mechanically deformed (compressed or stretched) by two patch-pipettes. Results: These cells had a resting potential (E0) of −37±3 mV and an input resistance of 514±11 MΩ. At intracellular pCa 7 (patch-pipette solution), compression of 2 or 3 μm shifted E0 from −36±7 to −17±3 mV, and to −10±2 mV. Compression by 2 or 3 μm induced a negative difference current (at −45 mV −0.06±0.02 and −0.20±0.04 nA, respectively) with a reversal potential (Erev) of approx. 0 mV. The currents were carried by Na+, K+ and Cs+ ions, and were blocked by application of 8 μM Gd3+. Stretch of 2 or 3 μm hyperpolarized E0 from −34±4 to −45±5, and to −61±7 mV and induced a positive difference current (at −45 mV: 0.04±0.02 and 0.18±0.03 nA) with an Erev close to 0 mV. Application of Gd3+ shifted E0 to potentials as negative as EK (−90±4 mV). Cell dialysis with 5 mM BAPTA (pCa 8) or 5 mM Ca2+/EGTA (pCa 6) had no influence on non-selective cation currents suggesting that Ca2+ dependent conductances are unlikely to contribute. Conclusion: Compression of the isolated cardiac fibroblast caused depolarization of the membrane by activating inward currents through a non-selective cation conductance (Gns). Stretch hyperpolarizes the fibroblast, however, not by Ca2+ activation of K+-conductance. Ion selectivity, Erev, and Gd3+-sensitivity of stretch suppressed currents suggest that stretch reduces Gns that is activated by compression. Ion transport, Stretch/m-e coupling, Vasoconstriction/dilation Time for primary review 23 days. 1 Introduction Fibroblasts are the most numerous non-myocyte cells of the heart [1]. They constitute a volume-fraction of 5–10% of the heart cell volume [2] and up to 75% in the sinus node [3]. Fibroblasts are involved in biochemical and structural changes during cardiac development and remodeling [4,5]. The possible contribution of fibroblasts to the cardiac electrophysiology is less clear. Microelectrode recordings from rat atrial multicellular tissue preparations indicate that fibroblasts have resting potential of −22±1.9 mV and input resistance of 0.51±0.01 GΩ [6,7]. The fibroblast membrane potential was shown to be modulated by mechanical stretch and pressure of the tissue [6–8], a property that may contribute to the intracardiac mechano-electrical feedback that increases the heart rate when the sinus node is stretched [8–19]. The changes in fibroblast membrane potential during the cardiac contraction have been called ‘mechanically induced potentials’ or MIPs [8]. MIP depolarization occurs during the force transient of the atrial tissue. It was speculated [11] that the surrounding contracting myocytes would compress the interposed fibroblast; what would lead to mechanical activation of non-selective cation channels [12] which was thought to depolarize the cell towards the reversal potential of approx. −5 mV. MIP hyperpolarization was observed when tissue was stretched by pre-load. It was interpreted [11]that the fibroblast would respond to stretch by Ca2+ influx through mechano-sensitive channels [13] and Ca2+ release from the endoplasmic reticulum [5], the resulting increase in the cytosolic Ca2+ concentration [Ca2+]c causing hyperpolarization by activation of K+-conductance. The above interpretations remain speculative because the respective currents have not yet been measured. Hence, we patch-clamped isolated atrial fibroblasts to analyze the ionic currents as they are induced by lateral stretch or compression. Lateral compression of the isolated cell caused MIP depolarization by activating inward currents through a non-selective cation conductance (Gns). We also can show that lateral stretch hyperpolarizes the fibroblast, however, not by Ca2+ activation of K+-conductance but by stretch-induced suppression of Gns. 2 Methods 2.1 Animals The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). Experiments were performed on hearts of adult rats of either sex weighing approximately 450 g. The animals were killed by decapitation. 2.2 Solutions Fibroblasts were perfused with (37 °C) physiological salt solution (PSS-Ko) containing (in mM) 150 NaCl, 5.4 KCl, 1.8 CaCl2, 1.2 MgCl2, 20 glucose, 5 HEPES/NaOH (pH 7.4). PSS-Cso had 5.4 mM KCl replaced by 5.4 mM CsCl. At the end of the experiments 8 μM GdCl3 was added to the PSS. Patch-pipette internal solution (Ki) contained (in mM) 140 KCl, 4.5 MgCl2, 4 Na2ATP, 10 HEPES/KOH (pH 7.4). ‘pCa 7 patch-pipette internal solution’ contained additional 5 μM EGTA. ‘pCa 6 patch-pipette internal solution’ was complemented with 4.5 mM CaCl2 and 5 mM EGTA (pH re-adjusted). ‘BAPTA patch-pipette internal solution’ was complemented with concentration 5 mM BAPTA (pCa=8). In some cases, 140 mM KCl was replaced by 140 mM CsCl in patch-pipette internal solution (Csi). 2.3 Preparation of atrial fibroblasts Rat atrial fibroblasts were acutely isolated by collagenase-dispersion followed by centrifugation [14]. Briefly, the isolated heart was perfused via a Langendorff cannula at 36 °C using a peristaltic pump. The initial (5 min) perfusion with Ca2+-free PSS was followed (15 min) by Ca2+-free PSS complemented with 0.1% collagenase (Worthington type II, Lakewood, NJ, USA) and 20 μM CaCl2. Finally, the atria were cut off and gently triturated to release the cells. From the supernatant, fibroblasts were separated by gentle centrifugation (two times centrifugation at 38×g for 1 min to collect cardiomyocytes on the bottom of the centrifuge tube, one time centrifugation of the supernatant at 300×g for 10 min to collect fibroblasts). Fibroblasts were identified by their electrophysiological properties. 2.4 Electrophysiology Patch-pipettes were pulled of thick-wall 2 mm diameter glass capillaries with a filament and were polished to a resistance of 2–3 MΩ. The pipettes were fixed to a Narishige hydraulic micromanipulator for whole-cell voltage clamp (PP), and to a Sutter MP285 manipulator (Sutter, Novato, CA, USA) for the mechanical deformations (SP, programmable stepping motor with 0.2 μm resolution). Whole-cell currents were recorded with a patch-clamp amplifier (RK 300, Biologic, Echirolle, France). Data were filtered at a cut-off frequency of 1000 Hz, digitized at 2000 Hz and stored on computer disk. A CED-1401 (Cambridge Instruments, Cambridge, UK) performed the A–D conversion, delivered the necessary analogue signals and controlled the timing of the experiment. No capacity or leakage compensation was applied. The I–V relations were obtained by 20 pulses of 140 ms (0.5 Hz) that started at a holding potential of −45 mV and went to 0, −100, −90, −80, −70, −60, −50, −40, −35, −30, −25, −20, −15, −10, −5, 0, 10, 20, 30, and 50 mV. The holding potential of −45 mV was used on the assumption that it were close to the resting potential of the fibroblast. The first step went to 0 mV to have a comparison with the currents during the 16th step that went also to 0 mV (possible modification of the cell be large membrane currents). Currents in response to trains of short 5 mV pulses (applied at −45 mV) were evaluated in terms of the membrane capacitance (time integral) and the access resistance (time constant divided by time integral) [15]. Membrane currents flowing at the end of the pulse (‘late current’ IL) were plotted versus the respective clamp step potential. The intercept of the resulting I–V curve with the voltage axis defined the zero current potential (E0) that corresponds to the resting potential of a non-clamped cell. From the IL before the mechanical stress and IL after mechanical stress, we plotted and calculated at −45 mV and at −90 mV for the compression of the cell ‘compression induced difference current’ (Ici) and for the stretch of the cell ‘stretch-induced difference current’ (Istr). 2.5 Mechanical deformation of cardiac fibroblasts Acutely isolated cardiac fibroblasts were 16–30 μm long, 10–20 μm wide and approx. 12 μm high. The fibroblasts were mechanically deformed between two patch-pipettes (PP and SP). The PP was used for whole-cell clamp and served as a fix point. The SP-cell attached pipette was laterally displaced in regard with the PP, thereby compressing or stretching the cell. We demonstrate the reaction of the fibroblast membrane to mechanical deformation, applied from the center of the cell. The increase in the distance PP–SI is termed ‘stretch’, the reduction is termed ‘compression’. The stretch–relaxation–compression experiment could be repeated on the same cell three times, on average. It was the aim of this study to compare the effects of reversible stretching and compression in the same cell. 2.6 Statistics For statistics, we used only experiments with complete protocol. We used 66 isolated cells (Table 1). Values are given as mean±S.D.; n denotes the number of cells from which the data were obtained. The mean values for the changes caused by compression or stretch were calculated from currents recorded from the same cell before and after the intervention. Significant differences were determined by analysis of variance (ANOVA) with the Bonferroni test as post-hoc test. Table 1 Electrophysiological properties of acutely isolated rat atrial fibroblasts under mechanical deformation and influence of bathing solutions and of patch-pipette solutions with different pCa Solution . Mechanical . ΔL . n . R . C . E0 . E0,ΔL . Ici or Istr at . Ici or Istr at . Erev . E0,ΔL,Gd . configurations . deformation . (μm) . . (MΩ) . (pF) . (mV) . (mV) . –45 mV (nA) . –90 mV (nA) . (mV) . (mV) . Ki/Ko, pCa 6 Compression 2 4 588±76 18±2 −34±5 −24±3 −0.03±0.01 −0.05±0.02 +5±3 −87±6 Compression 3 4 577±80 19±2 −35±5 −10±3 −0.18±0.05*** −0.33±0.04*** +11±2 −80±5 Compression 4 5 539±96 18±2 −33±6 −5±2 −0.34±0.09+++‡ −0.63±0.11+++‡ +16±3 −85±6 Stretch 2 4 501±57 18±2 −35±6 −43±6 0.05±0.02 0.09±0.02 0±2 −97±5 Stretch 3 4 547±85 16±2 −35±5 −52±3 0.12±0.04*** 0.20±0.05** −6±1 −98±4 Stretch 4 4 585±48 18±2 −33±3 −82±8 0.37±0.03+++†† 1.35±0.24+++†† −10±2 −95±4 Ki/Ko, pCa 7 Compression 2 4 486±23 16±3 −36±7 −17±3 −0.06±0.02 −0.08±0.02 0±2 −88±6 Compression 3 4 492±34 16±2 −36±8 −10±2 −0.20±0.04*** −0.36±0.05*** +5±2 −90±4 Stretch 2 5 584±44 18±2 −30±4 −45±5 0.04±0.02 0.06±0.02 −3±3 −86±4 Stretch 3 4 576±39 18±3 −34±4 −61±7 0.18±0.03*** 0.26±0.11*** −8±3 −88±5 Csi/Cso, pCa 7 Compression 3 4 460±28 18±3 −33±8 −8±4 −0.25±0.08 −0.41±0.10 0±3 −85±4 Stretch 2 4 486±32 19±2 −35±5 −45±4 0.06±0.02 0.10±0.03 0±2 −88±5 Ki/Ko, pCa 8 Compression 2 4 496±31 16±2 −25±4 −15±3 −0.04±0.01 −0.07±0.02 0±2 −75±4 Compression 3 3 523±96 18±2 −25±3 −5±3 −0.20±0.09*** −0.33±0.11*** 5±3 −70±5 Stretch 2 5 584±44 20±3 −27±3 −40±5 0.05±0.02 0.07±0.03 5±2 −82±3 Stretch 3 4 575±38 21±3 −25±4 −60±6 0.16±0.03*** 0.28±0.08*** 5±3 −98±4 Solution . Mechanical . ΔL . n . R . C . E0 . E0,ΔL . Ici or Istr at . Ici or Istr at . Erev . E0,ΔL,Gd . configurations . deformation . (μm) . . (MΩ) . (pF) . (mV) . (mV) . –45 mV (nA) . –90 mV (nA) . (mV) . (mV) . Ki/Ko, pCa 6 Compression 2 4 588±76 18±2 −34±5 −24±3 −0.03±0.01 −0.05±0.02 +5±3 −87±6 Compression 3 4 577±80 19±2 −35±5 −10±3 −0.18±0.05*** −0.33±0.04*** +11±2 −80±5 Compression 4 5 539±96 18±2 −33±6 −5±2 −0.34±0.09+++‡ −0.63±0.11+++‡ +16±3 −85±6 Stretch 2 4 501±57 18±2 −35±6 −43±6 0.05±0.02 0.09±0.02 0±2 −97±5 Stretch 3 4 547±85 16±2 −35±5 −52±3 0.12±0.04*** 0.20±0.05** −6±1 −98±4 Stretch 4 4 585±48 18±2 −33±3 −82±8 0.37±0.03+++†† 1.35±0.24+++†† −10±2 −95±4 Ki/Ko, pCa 7 Compression 2 4 486±23 16±3 −36±7 −17±3 −0.06±0.02 −0.08±0.02 0±2 −88±6 Compression 3 4 492±34 16±2 −36±8 −10±2 −0.20±0.04*** −0.36±0.05*** +5±2 −90±4 Stretch 2 5 584±44 18±2 −30±4 −45±5 0.04±0.02 0.06±0.02 −3±3 −86±4 Stretch 3 4 576±39 18±3 −34±4 −61±7 0.18±0.03*** 0.26±0.11*** −8±3 −88±5 Csi/Cso, pCa 7 Compression 3 4 460±28 18±3 −33±8 −8±4 −0.25±0.08 −0.41±0.10 0±3 −85±4 Stretch 2 4 486±32 19±2 −35±5 −45±4 0.06±0.02 0.10±0.03 0±2 −88±5 Ki/Ko, pCa 8 Compression 2 4 496±31 16±2 −25±4 −15±3 −0.04±0.01 −0.07±0.02 0±2 −75±4 Compression 3 3 523±96 18±2 −25±3 −5±3 −0.20±0.09*** −0.33±0.11*** 5±3 −70±5 Stretch 2 5 584±44 20±3 −27±3 −40±5 0.05±0.02 0.07±0.03 5±2 −82±3 Stretch 3 4 575±38 21±3 −25±4 −60±6 0.16±0.03*** 0.28±0.08*** 5±3 −98±4 Mechanical deformation: compression or stretch of cell. ΔL, distance of lateral compression or stretch; n, number of experiments; R, input membrane resistance; C, membrane capacity; E0 intercept of the I–V curve with voltage axis (zero current potential corresponding to resting potential of non-clamped cell); E0,ΔL, change of E0 after application of ΔL; Ici or Istr, ‘compression induced difference current’ or ‘stretch-induced difference current’, respectively; Erev, reversal potential; E0,ΔL,Gd, change of E0 during mechanical deformation of ΔL in presence of Gd3+. Values of R, C, E0, E0,ΔL,Gd were not significantly different for each type and value of the mechanical deformation. 3 μm vs. 2 μm: (*)P<0.05; **P<0.01; ***P<0.001; 4 μm vs. 2 μm (+)P<0.05; +++P<0.001; 4 μm vs. 3 μm; †P<0.05, ‡P<0.01, ††P<0.001 Open in new tab Table 1 Electrophysiological properties of acutely isolated rat atrial fibroblasts under mechanical deformation and influence of bathing solutions and of patch-pipette solutions with different pCa Solution . Mechanical . ΔL . n . R . C . E0 . E0,ΔL . Ici or Istr at . Ici or Istr at . Erev . E0,ΔL,Gd . configurations . deformation . (μm) . . (MΩ) . (pF) . (mV) . (mV) . –45 mV (nA) . –90 mV (nA) . (mV) . (mV) . Ki/Ko, pCa 6 Compression 2 4 588±76 18±2 −34±5 −24±3 −0.03±0.01 −0.05±0.02 +5±3 −87±6 Compression 3 4 577±80 19±2 −35±5 −10±3 −0.18±0.05*** −0.33±0.04*** +11±2 −80±5 Compression 4 5 539±96 18±2 −33±6 −5±2 −0.34±0.09+++‡ −0.63±0.11+++‡ +16±3 −85±6 Stretch 2 4 501±57 18±2 −35±6 −43±6 0.05±0.02 0.09±0.02 0±2 −97±5 Stretch 3 4 547±85 16±2 −35±5 −52±3 0.12±0.04*** 0.20±0.05** −6±1 −98±4 Stretch 4 4 585±48 18±2 −33±3 −82±8 0.37±0.03+++†† 1.35±0.24+++†† −10±2 −95±4 Ki/Ko, pCa 7 Compression 2 4 486±23 16±3 −36±7 −17±3 −0.06±0.02 −0.08±0.02 0±2 −88±6 Compression 3 4 492±34 16±2 −36±8 −10±2 −0.20±0.04*** −0.36±0.05*** +5±2 −90±4 Stretch 2 5 584±44 18±2 −30±4 −45±5 0.04±0.02 0.06±0.02 −3±3 −86±4 Stretch 3 4 576±39 18±3 −34±4 −61±7 0.18±0.03*** 0.26±0.11*** −8±3 −88±5 Csi/Cso, pCa 7 Compression 3 4 460±28 18±3 −33±8 −8±4 −0.25±0.08 −0.41±0.10 0±3 −85±4 Stretch 2 4 486±32 19±2 −35±5 −45±4 0.06±0.02 0.10±0.03 0±2 −88±5 Ki/Ko, pCa 8 Compression 2 4 496±31 16±2 −25±4 −15±3 −0.04±0.01 −0.07±0.02 0±2 −75±4 Compression 3 3 523±96 18±2 −25±3 −5±3 −0.20±0.09*** −0.33±0.11*** 5±3 −70±5 Stretch 2 5 584±44 20±3 −27±3 −40±5 0.05±0.02 0.07±0.03 5±2 −82±3 Stretch 3 4 575±38 21±3 −25±4 −60±6 0.16±0.03*** 0.28±0.08*** 5±3 −98±4 Solution . Mechanical . ΔL . n . R . C . E0 . E0,ΔL . Ici or Istr at . Ici or Istr at . Erev . E0,ΔL,Gd . configurations . deformation . (μm) . . (MΩ) . (pF) . (mV) . (mV) . –45 mV (nA) . –90 mV (nA) . (mV) . (mV) . Ki/Ko, pCa 6 Compression 2 4 588±76 18±2 −34±5 −24±3 −0.03±0.01 −0.05±0.02 +5±3 −87±6 Compression 3 4 577±80 19±2 −35±5 −10±3 −0.18±0.05*** −0.33±0.04*** +11±2 −80±5 Compression 4 5 539±96 18±2 −33±6 −5±2 −0.34±0.09+++‡ −0.63±0.11+++‡ +16±3 −85±6 Stretch 2 4 501±57 18±2 −35±6 −43±6 0.05±0.02 0.09±0.02 0±2 −97±5 Stretch 3 4 547±85 16±2 −35±5 −52±3 0.12±0.04*** 0.20±0.05** −6±1 −98±4 Stretch 4 4 585±48 18±2 −33±3 −82±8 0.37±0.03+++†† 1.35±0.24+++†† −10±2 −95±4 Ki/Ko, pCa 7 Compression 2 4 486±23 16±3 −36±7 −17±3 −0.06±0.02 −0.08±0.02 0±2 −88±6 Compression 3 4 492±34 16±2 −36±8 −10±2 −0.20±0.04*** −0.36±0.05*** +5±2 −90±4 Stretch 2 5 584±44 18±2 −30±4 −45±5 0.04±0.02 0.06±0.02 −3±3 −86±4 Stretch 3 4 576±39 18±3 −34±4 −61±7 0.18±0.03*** 0.26±0.11*** −8±3 −88±5 Csi/Cso, pCa 7 Compression 3 4 460±28 18±3 −33±8 −8±4 −0.25±0.08 −0.41±0.10 0±3 −85±4 Stretch 2 4 486±32 19±2 −35±5 −45±4 0.06±0.02 0.10±0.03 0±2 −88±5 Ki/Ko, pCa 8 Compression 2 4 496±31 16±2 −25±4 −15±3 −0.04±0.01 −0.07±0.02 0±2 −75±4 Compression 3 3 523±96 18±2 −25±3 −5±3 −0.20±0.09*** −0.33±0.11*** 5±3 −70±5 Stretch 2 5 584±44 20±3 −27±3 −40±5 0.05±0.02 0.07±0.03 5±2 −82±3 Stretch 3 4 575±38 21±3 −25±4 −60±6 0.16±0.03*** 0.28±0.08*** 5±3 −98±4 Mechanical deformation: compression or stretch of cell. ΔL, distance of lateral compression or stretch; n, number of experiments; R, input membrane resistance; C, membrane capacity; E0 intercept of the I–V curve with voltage axis (zero current potential corresponding to resting potential of non-clamped cell); E0,ΔL, change of E0 after application of ΔL; Ici or Istr, ‘compression induced difference current’ or ‘stretch-induced difference current’, respectively; Erev, reversal potential; E0,ΔL,Gd, change of E0 during mechanical deformation of ΔL in presence of Gd3+. Values of R, C, E0, E0,ΔL,Gd were not significantly different for each type and value of the mechanical deformation. 3 μm vs. 2 μm: (*)P<0.05; **P<0.01; ***P<0.001; 4 μm vs. 2 μm (+)P<0.05; +++P<0.001; 4 μm vs. 3 μm; †P<0.05, ‡P<0.01, ††P<0.001 Open in new tab 3 Results 3.1 Currents in non-deformed fibroblasts Isolated fibroblasts had a membrane capacity of 18±3 pF (n=36) and a membrane resistance Rm of 514±11 MΩ (n=50). The amplitude of the currents during both depolarizing and hyperpolarizing clamp steps are demonstrated in Fig. 1A. We assume that the analyzed cells were fibroblasts because: (a) fibroblasts comprise 90% of the cardiac non-myocytes [1]; and (b) these cells did not show membrane currents characteristic for smooth muscle or endothelial cells [16,17]. Depolarizing clamp steps induced outward currents that peaked within approx. 20 ms and then slowly decayed. There were no time dependent Ca2+ inward currents that are typical for myocytes from coronary arteries [18]. Upon repolarization, the current ‘jumped’ to the holding current; inward tail currents typical for the endothelial cells were absent [19]. Thus, the membrane currents support the assumption that we analyzed neither coronary smooth muscle nor endothelial cells. Fig. 1 Open in new tabDownload slide Examples of electrophysiological characteristics of acute isolated fibroblast during ‘resting compression’ by patch-pipettes. (A) Whole-cell currents from cardiac fibroblast in control. Starting from a holding potential of −45 mV, pulses of 140 ms duration were applied at 0.5 Hz. Clamp step potentials went to 0, −100, −90, −80, −70, −60, −50, −40, −35, −30, −25, −20, −15, −10, −5, 0, 10, 20, 30 and 50. (B) Whole-cell currents after addition of 8 μM Gd3+. (C) The I–V curves of the IL in control (open triangles, E0 −40 mV) and after application of 8 μM Gd3+ (filled triangles, E0−100 mV). Note: pCa 7, Ki/Ko solution configuration. Fig. 1 Open in new tabDownload slide Examples of electrophysiological characteristics of acute isolated fibroblast during ‘resting compression’ by patch-pipettes. (A) Whole-cell currents from cardiac fibroblast in control. Starting from a holding potential of −45 mV, pulses of 140 ms duration were applied at 0.5 Hz. Clamp step potentials went to 0, −100, −90, −80, −70, −60, −50, −40, −35, −30, −25, −20, −15, −10, −5, 0, 10, 20, 30 and 50. (B) Whole-cell currents after addition of 8 μM Gd3+. (C) The I–V curves of the IL in control (open triangles, E0 −40 mV) and after application of 8 μM Gd3+ (filled triangles, E0−100 mV). Note: pCa 7, Ki/Ko solution configuration. The experiment was finished by addition of 8 μM Gd3+ to the superfusing PSS. Gd3+ shifted the holding current into the positive direction (beginning of the traces in Fig. 1B) and decreased current amplitudes during depolarizing and hyperpolarizing clamp steps (Fig. 1B). The I–V curves of the late currents intersected the zero current axis at different values of E0 (mean, −37±3 mV, n=50). Fig. 1C (open triangles) demonstrates the typical example of E0=−40 mV, a value that reflects the resting potential of current-clamped cells. We were concerned that the different values of E0 had resulted from a ‘resting compression’ of the cell by the two patch-pipettes. Without artificial compression or stretch, 8 μM Gd3+ shifted the intercept of the I–V curve (IL) with the voltage axis leftward indicating hyperpolarization, and E0 shifted from −34±4 to −98±5 mV (n=10). Fig. 1C (open triangles) demonstrates the effect, when two patch-pipettes are attached to the cell: PP in whole-cell configuration and SP in cell-attached mode. In this case E0 was −40 mV. Addition of 8 μM Gd3+ (Fig. 1C filled triangles) shifted E0 during the first 7±1 min to −92±4 mV. During the following time (15 min) Gd3+ further hyperpolarized E0 toward values more negative than −100 mV (−120 mV by linear approximation). Fig. 1 may suggest a ‘resting compression’ by the two patch-pipettes even in absence of lateral displacement. This possibility was tested by comparing currents measured with a single patch-pipette. Under these conditions, E0 was −35±5 mV (n=8). Addition of 8 μM Gd3+ shifted E0 to −90±5 mV during the first 7±1 min (n=8). Since these values did not differ from those measured with two patch-pipettes we postulate that activation of Gns by the second cell attached patch-pipette was negligible. The results suggest that a Gd3+-sensitive non-selective membrane conductance Gns is active in atrial fibroblasts under ‘normal recording conditions’, and that this Gns moves the resting potential away from the EK. 3.2 Compression increases the membrane conductance Compression shifted the holding current at −45 mV to the negative direction (beginning of the traces in Fig. 2B in comparison with the beginning of the traces in Fig. 2A). It increased the currents during the depolarizing clamp steps to larger amplitudes without changing their time course. At negative potentials, the currents were more negative and at positive potentials more positive than their respective controls. That is, compression increased the membrane conductance. The experiment was finished by addition of 8 μM Gd3+ to the superfusing PSS that blocked the compression-induced shift of the holding current and decreased currents amplitudes during depolarizing and hyperpolarizing clamp steps (Fig. 2C). Fig. 2 Open in new tabDownload slide Whole-cell currents from cardiac fibroblasts. (A) At control. (B) During 2 μm of compression. (C) After addition of 8 μM Gd3+. Note: The pulse conditions are similar to Fig. 1A; pCa 7; Ki/Ko solution configuration. Fig. 2 Open in new tabDownload slide Whole-cell currents from cardiac fibroblasts. (A) At control. (B) During 2 μm of compression. (C) After addition of 8 μM Gd3+. Note: The pulse conditions are similar to Fig. 1A; pCa 7; Ki/Ko solution configuration. The changes in membrane currents continued as long as the compression was sustained, i.e. there were no signs of adaptation (tested for up to 15 min). The effect of compression was reversible, i.e. after the end of mechanical deformation the currents returned within less than 0.5 s to the control (Fig. 3). Fig. 3 Open in new tabDownload slide Modulation of net membrane compression-induced current. (A) On-line pen recordings. (a,b,c) depict the current during compression by 2, 3, 4 μm, respectively. (d,e) demonstrate the offset of the mechanical deformation of the cell. (B) Current–voltage relation of IL (empty triangles) before compression by 2 μm, which depolarized E0 from −20 to −10 mV and increased IL (filled triangles). In this case, Ici was −0.04 nA (at −45 mV) and −0.1 nA (at −90 mV). Offset of the compression results to completely reversibility of this compression induced effect IL (filled circles). Note: pCa 7, Ki/Ko solution configuration. Fig. 3 Open in new tabDownload slide Modulation of net membrane compression-induced current. (A) On-line pen recordings. (a,b,c) depict the current during compression by 2, 3, 4 μm, respectively. (d,e) demonstrate the offset of the mechanical deformation of the cell. (B) Current–voltage relation of IL (empty triangles) before compression by 2 μm, which depolarized E0 from −20 to −10 mV and increased IL (filled triangles). In this case, Ici was −0.04 nA (at −45 mV) and −0.1 nA (at −90 mV). Offset of the compression results to completely reversibility of this compression induced effect IL (filled circles). Note: pCa 7, Ki/Ko solution configuration. Fig. 4A1 demonstrates a typical example of the increase in net membrane currents during compression. In general, compression depolarized E0 (Table 1, pCa 7, Ki/Ko). Starting from −36±7 mV, 2 μm compression shifted E0 to −17±3 mV, 3 μm compression to −10±2 mV. Ici reversed at Erev 0±2 and +5±2 mV, respectively. Increase in the compression increased Ici. Gd3+ reduced the compression induced currents; when the Gd3+ effects had become steady (after approx. 10 min), Gd3+ had shifted E0 to −88±6 mV and −90±4 mV for compression by 2 and 3 μm, respectively. The current blocked by Gd3+ rectified outwardly and reversed at 0 mV (Fig. 4B2). In the presence of 8 μM Gd3+, compression could no longer modify the membrane currents. Fig. 4 Open in new tabDownload slide Mechanical compression increases the net membrane conductance. (A1) Control (empty triangles, E0=−46 mV), 2 μm compression (filled circles, E0=−16 mV) and 3 μm compression (filled squares, E0=−12 mV). (A2) Compression-induced current (subtracting triangles from squares, Erev=+9 mV). (B1) 7 min after superfusion with 8 μM Gd3+ (empty triangles, E0=−92 mV) in comparison with compression alone (circles, 3 μm compression). (B2) Gd3+ sensitive difference current (Erev=0 mV). Note: pCa 7, Ki/Ko solution configuration. Fig. 4 Open in new tabDownload slide Mechanical compression increases the net membrane conductance. (A1) Control (empty triangles, E0=−46 mV), 2 μm compression (filled circles, E0=−16 mV) and 3 μm compression (filled squares, E0=−12 mV). (A2) Compression-induced current (subtracting triangles from squares, Erev=+9 mV). (B1) 7 min after superfusion with 8 μM Gd3+ (empty triangles, E0=−92 mV) in comparison with compression alone (circles, 3 μm compression). (B2) Gd3+ sensitive difference current (Erev=0 mV). Note: pCa 7, Ki/Ko solution configuration. Table 1 demonstrates changes of E0 and Ici with the patch-pipette internal solution pCa 6, pCa 7 and pCa 8 in Ki/Ko solution configuration. For all different pCa values, a similar degree of compression-induced a similar depolarization and a similar increase Ici. This suggests that compression-induced depolarization and Ici are independent of the pCa. 3.3 Compression increases a non-selective cation conductance Gns In some experiments, we substituted K+ by Cs+ ions in both PSS and internal patch-pipette solution (Csi/Cso solution configuration). After 5 min of whole-cell access, the E0 was −33±8 mV (Table 1, pCa 7, Csi/Cso; n.s. vs. pCa7 Ki/Ko). With Cs+ as charge carrier solution (Csi/Cso solution configuration), the control current (IL) was somewhat larger than its counterpart in Ki/Ko solution. With Cs+, a 3 μm compression depolarized E0 from −33±8 to −8±4 mV. At −45 mV, compression by 3 μm induced a difference current Ici of −0.25±0.08 nA. The compression induced difference current reversed the polarity at 0±3 mV, and rectified outwardly. Addition of 8 μM Gd3+ hyperpolarized E0 to −85±4 mV during compression and suppressed Ici. The data suggest that Cs+ ions can carry the current through the compression-activated Gd3+-sensitive conductance. 3.4 Stretch reduces Gns Fig. 5 shows membrane currents recorded at control, during 2 μm lateral stretch and that 7 min after addition of 8 μM GdCl3 (applied during continuous stretch). Stretch shifted the holding current at −45 mV to more positive values (beginning of the traces in Fig. 5B). Stretch reduced the amplitude of the currents during the pulses during both, depolarizing and at hyperpolarizing clamp steps, suggesting that stretch reduced the membrane conductance (Fig. 5B). The time course of the currents did not change significantly. The experiment was finished by addition of 8 μM Gd3+ to the superfusing PSS. The currents that were reduced by stretch could be further reduced by Gd+3 (Fig. 5C). Fig. 5 Open in new tabDownload slide Whole-cell currents from cardiac fibroblasts. (A) At control. (B) During 2 μm of stretch. (C) After addition of 8 μM Gd3+. The pulses conditions are similar to that in Fig. 1A. Note: pCa 7, Ki/Ko solution configuration. Fig. 5 Open in new tabDownload slide Whole-cell currents from cardiac fibroblasts. (A) At control. (B) During 2 μm of stretch. (C) After addition of 8 μM Gd3+. The pulses conditions are similar to that in Fig. 1A. Note: pCa 7, Ki/Ko solution configuration. The changes in membrane currents continued as long as stretch was sustained, i.e. there were no signs of adaptation (tested up to 15 min). The effect of stretch was reversible, i.e. after the end of mechanical deformation the currents returned within less than 1 s to the control (Fig. 6). Fig. 6 Open in new tabDownload slide Reversibility of stretch induced currents. (A) On-line pen recordings (a,b,c), depict the current during stretch by 2, 3, 4 μm, respectively; (d) demonstrates the offset of the mechanical deformation of the cell. (B1) Current–voltage relation of IL before stretch (empty triangles) and during stretch by 2 μm, which hyperpolarized E0 from −35 to −60 mV and decreased IL (filled triangles). In this case, Istr was 0.02 nA (at −45 mV) and 0.033 nA (at −90 mV). Offset of the stretch results to completely reversibility of this stretch-induced effect IL (filled circles). (B2) IL before compression (empty triangles) and during compression by 2 μm, which depolarized E0 from −35 to −28 mV and increased IL (filled triangles). In this case, Ici was −0.02 nA (at −45 mV) and −0.04 nA (at −90 mV). Note: pCa 7, Ki/Ko solution configuration. Fig. 6 Open in new tabDownload slide Reversibility of stretch induced currents. (A) On-line pen recordings (a,b,c), depict the current during stretch by 2, 3, 4 μm, respectively; (d) demonstrates the offset of the mechanical deformation of the cell. (B1) Current–voltage relation of IL before stretch (empty triangles) and during stretch by 2 μm, which hyperpolarized E0 from −35 to −60 mV and decreased IL (filled triangles). In this case, Istr was 0.02 nA (at −45 mV) and 0.033 nA (at −90 mV). Offset of the stretch results to completely reversibility of this stretch-induced effect IL (filled circles). (B2) IL before compression (empty triangles) and during compression by 2 μm, which depolarized E0 from −35 to −28 mV and increased IL (filled triangles). In this case, Ici was −0.02 nA (at −45 mV) and −0.04 nA (at −90 mV). Note: pCa 7, Ki/Ko solution configuration. Fig. 7 demonstrates a typical example of the stretch-induced changes in net membrane currents. With a physiological K+ gradient, stretch hyperpolarized E0 (Table 1, pCa 7, Ki/Ko). Starting from −30±4 mV, a 2 μm stretch changed E0 to −45±5 mV, and a 3 μm stretch to −61±7 mV with Erev=−3±3 and −8±3 mV, respectively. Application of stretch induced a positive Istr or suppressed Ici. When Gd3+ had been applied for 8 min, E0 was shifted to −86±4 mV and −88±5 mV, respectively. Addition of 8 μM Gd3+ reduced the membrane conductance below the reduction obtained by mechanical stretch. The I–V curve values recorded under stretch application in the presence of Gd3+ had a reversal potential of 0 mV and also rectified outwardly. Fig. 7 Open in new tabDownload slide Mechanical stretch suppresses the net membrane conductance. (A1) Control (empty triangles) to 2 μm stretch (filed circles, E0 −70 mV) and to stretch plus 8 μM Gd3+ (8 min, filled squares; E0 linearly extrapolated to −120 mV). (A2) Stretch reduced difference current. Note: pCa 7, Ki/Ko solution configuration. Fig. 7 Open in new tabDownload slide Mechanical stretch suppresses the net membrane conductance. (A1) Control (empty triangles) to 2 μm stretch (filed circles, E0 −70 mV) and to stretch plus 8 μM Gd3+ (8 min, filled squares; E0 linearly extrapolated to −120 mV). (A2) Stretch reduced difference current. Note: pCa 7, Ki/Ko solution configuration. After substitution of K+ by Cs+ ions, stretch hyperpolarized E0 from −33±8 to −56±4 mV (Table 1, pCa 7, Csi/Cso). In summary, the results suggest that stretch reduces a non-selective cation conductance Gns. This conclusion is in line with the result that superfusion of 8 μm Gd3+ under continuous application of stretch reduced both conductance at negative potentials and conductance at positive potentials, and that cells pre-treated with 8 μM Gd3+ were nearly insensitive to stretch. 3.5 Does the Ca2+ activated K+-conductance GK,Ca contribute to mechano-sensitivity? This possibility was tested by dialyzing into fibroblasts either 5 mM BAPTA (pCa 8, Ki/Ko solution configuration) or 5 mM Ca2+/EGTA (pCa 6, Ki/Ko solution configuration). Data acquisition started 5 min after rupture of the patch. In general, dialysis of BAPTA (Table 1, pCa 8, Ki/Ko) did not prevent the effects of compression and stretch. Ca2+/EGTA mixture was dialyzed into the cells to augment the putative Ca2+ activation of GK by increasing cytosolic [Ca2+]c and by enhancing the Ca2+ filling of intracellular stores. Dialysis of Ca2+/EGTA mixture also did not prevent the effects of compression and stretch. The properties of E0, and currents of cells dialyzed with internal patch-pipette solution, containing 5 mM BAPTA resembled those obtained with 5 μM EGTA and Ca2+/EGTA mixture. On average, no significant differences of E0, Istr could be established between cells dialyzed with pCa 6 patch-pipette solution BAPTA and pCa 7 control solutions. This similarity was true for the non-deformed control cells, and for the values measured during compression and stretch. Hence, present results do not support the idea that the stretch-induced hyperpolarization of E0 is mediated via a Ca2+ activated conductance. 4 Discussion The present results confirm the mechano-sensitivity of the membrane potential of cardiac fibroblasts on the single cell level: compression depolarized and stretch hyperpolarized the zero current potential E0 as it was reported for fibroblasts in multicellular atrial tissue [11,21,22]. The properties of the compression induced membrane currents in fibroblasts closely resemble the properties of currents through stretch-activated non-selective cation channels [12]: the reversal potential was close to 0 mV, the current could be carried by Na+, K+ and Cs+. Furthermore, the compression activated conductance followed an outwardly rectifying voltage dependence, and it was inhibited by 8 μM Gd3+[23]. The following evidence argues that the current induced by lateral compression or stretch is caused by activation or deactivation of ion channels [20] and not by non-specific leakage: (1) In the cell attached mode, the seal resistance remained stable and did not change (n=10) with the compression or stretch (1.9±0.3 GΩ before and 1.9±0.4 GΩ after deformation). Only strong stretch (>35%) or strong compression (>20%) reduced the seal resistance. (2) The compression- or stretch-effects were reversible upon re-positioning the patch-pipette. (3) The voltage dependence of the compression-induced current was outwardly rectifying and not linear as one would expect from leakage. (4) The compression induced current was blocked by 8 μM Gd3+. (5) When Gd3+ was washed out by perfusion with PSS, we recorded a reaction to compression, which was similar to that we recorded before application of Gd+3. The observed hyperpolarization and depolarization can be modeled with a minimum of three current components: (1) a non-selective cation current through Gns; (2) a K+ current through a Gd3+- and mechano-insensitive potassium conductance GK; and (3) a current IP due to electrogenic Na+-pumping. With EK=−90 mV (Nernst potential) and Ens=0 mV (reversal potential for the non-selective cation current Ins) we write (1) With Eq. (1) our results suggest that approx. 85% of total conductance at potentials negative to E0 is due to the Gd3+ sensitive Gns if the fibroblast is not mechanically deformed. Eq. (1) attributes the compression-induced depolarization to the increase in Gns that shifts E0 towards Erev=0 mV. Similarly, Eq. (1) models the stretch-induced hyperpolarization as a shift of E0 towards EK=−90 mV caused by a reduced contribution of Gns. Application of Eq. (1) to the Gd3+ data faces the problem that a Gd3+ insensitive GK was observed not only with K+ ions but also with Cs+-ions and Na+ ions as charge carrier. We interpret that either Gd3+ block of outward currents through Gns was incomplete, or that small additional conductance components operate in parallel to GK in the fibroblast membrane. IP due to electrogenic sodium pumping was incorporated in Eq. (1) to account for the result that E0 could be more negative than EK. The hyperpolarization beyond −100 mV was usually a transient phenomenon, as if preceding influx Na+ through compression activated Gns had stimulated IP. Eq. (1) indicates that hyperpolarization increases in proportion to −IP/G = −IP/{Gns+GK} (in this case G is the slope conductance at E0). In the presence of Gd3+, E0=−120 mV requires a pump current of 1.2 pA. We extrapolate that a similar IP would add −11 mV to E0 during stretch, −7 mV to control E0, and −3 mV during compression. Thus, −4 mV=−11 mV−(−7) mV of the MIP hyperpolarization by stretch may be attributed to IP. The observed activation of Gns by compression supports the hypothesis that fibroblasts generate depolarizing MIPs when they are squeezed by the surrounding atrial myocytes [11,22]. How mechanical compression is translated into channel activation is not yet clear. The Gd3+ sensitivity of the effect suggests involvement of stretch-activated channels, however, it does not prove it since Gd3+ blocks a variety of different ion channels [24]. The present experiments tested the suggestion that the stretch-induced hyperpolarization were caused by a Ca2+ activated K conductance (GK,Ca). It was postulated that [11]: (1) stretch activated channels mediate Ca2+ influx [12,25], (2) cardiac fibroblasts can release Ca2+ from intracellular stores [5,25]; and (3) skin or gingival fibroblasts bear Ca2+ activated K+ channels [26]. The present results do not support the above hypothesis because (1) dialysis of 5 mM BAPTA did not modify resting, mechano-sensitive or Gd3+-insensitive currents; and (2) the stretch-induced hyperpolarization went along with a reduced slope conductance at potentials negative to E0 and not with an increased slope conductance at potentials negative to E0 (activated GK,Ca). Although the results are clear, they do not definitely exclude the postulated GK,Ca activation because cell dialysis may have washed out proteins involved in the Ca2+ activation process (e.g. calmodulin). It is still unclear how mechanical deformation translates into channel activation. One model postulates that the mechanical energy is transferred via changes in the tension of the lipid bilayer. In this model the tension increases with the radius of the curvature of the cell membrane [27,28]. When applied to the present study, the lateral compression that increases the cell radius (and lipid tension) in one place and would simultaneously reduce these parameter at an other place of the cell because the cytosol is incompressible. Hence, this hypothesis has problems to explain how lateral compression can activate and stretch can deactivate the same conductance Gns. Alternatively, the cytoskeleton has been postulated to translate mechanical energy from the place of deformation to the channel protein [27]. To explain the above contradiction one should postulate that the cytoskeleton translates the exogenous energy of compression and stretch with asymmetry. Perhaps, this asymmetry was induced by the two glass tools, it is known that pressure disrupts actin stress fibers and depolymerizes intermediate filaments in various cell types [29]. This may happen locally, when the glass tools are applied, the pressure is increased when the cell is compressed and decreased when the cell is stretched. This interpretation is supported by recent findings in fibroblasts from multicellular preparations, where disruption of cytoskeletal proteins depolarized the fibroblast membrane and stabilization of the cytoskeleton hyperpolarized the fibroblast membrane [22]. The similarity between the present single cell results and the MIPs of multicellular atrial trabeculae suggests that the asymmetrical response to mechanical deformation could also be inherent property of the native fibroblast. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (Tr 02-A3 and Is 17-2) and the Alexander von Humboldt-Stiftung (MPF). We thank Dr Fred Sachs, Department of Biophysics, SUNY, Buffalo, USA, for critical reading and helpful discussion of the manuscript. References [1] Eghbali M Czaja M.J Zeydel M et al. Collagen chain mRNAs in isolated heart cells from young and adult rats J Mol Cell Cardiol 1988 20 267 276 Google Scholar Crossref Search ADS PubMed WorldCat [2] Anversa P Olivetti G Melissari M Loud A.V Stereological measurement of cellular and subcellular hypertrophy and hyperplasia in the papillary muscle of adult rat J Mol Cell Cardiol 1980 12 781 795 Google Scholar Crossref Search ADS PubMed WorldCat [3] Shiraishi I Takamatsu T Mimikawa T Onouchi Z Fujita S Quantitative histological analysis of the human sinoatrial node during growth and aging Circulation 1992 85 2176 2184 Google Scholar Crossref Search ADS PubMed WorldCat [4] Dostal D.E Baker K.M The cardiac renin–angiotensin system—Conceptual, or a regulator of cardiac function? Circ Res 1999 85 643 650 Google Scholar Crossref Search ADS PubMed WorldCat [5] Meszaros J.G Gonzalez A.M Endo-Mochizuki Y et al. Identification of G protein-coupled signaling pathways in cardiac fibroblasts: cross talk between G(q) and G(s) Am J Physiol 2000 278 C154 C162 OpenURL Placeholder Text WorldCat [6] Kiseleva I Kamkin A Pylaev A et al. Electrophysiological properties of mechanosensitive atrial fibroblasts from chronic infarcted rat heart J Mol Cell Cardiol 1998 30 1083 1093 Google Scholar Crossref Search ADS PubMed WorldCat [7] Kamkin A Kiseleva I Wagner K.D et al. A possible role for atrial fibroblasts in postinfarction bradycardia Am J Physiol 2002 282 H842 H849 OpenURL Placeholder Text WorldCat [8] Kohl P Kamkin A.G Kiseleva I.S Noble D Mechanosensitive fibroblasts in the sino-atrial node region of rat heart: interaction with cardiomyocytes and possible role Exp Physiol 1994 79 943 956 Google Scholar Crossref Search ADS PubMed WorldCat [9] Nazir S.A Lab M.J Mechanoelectric feedback and atrial arrhythmias Cardiovasc Res 1996 32 52 61 Google Scholar Crossref Search ADS PubMed WorldCat [10] Lab M.J Mechanoelectric feedback (transduction) in heart: Concepts and implications Cardiovasc Res 1996 32 3 14 Google Scholar Crossref Search ADS PubMed WorldCat [11] Kiseleva I Kamkin A Kohl P Lab M.J Calcium and mechanically induced potentials in fibroblasts of rat atrium Cardiovasc Res 1996 32 98 111 Google Scholar Crossref Search ADS PubMed WorldCat [12] Hu H Sachs F Stretch-activated ion channels in the heart J Mol Cell Cardiol 1997 29 1511 1523 Google Scholar Crossref Search ADS PubMed WorldCat [13] Glogauer M Ferrier J McCulloch C.A.G Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts Am J Physiol 1995 269 C1093 C1104 Google Scholar PubMed OpenURL Placeholder Text WorldCat [14] Gallagher A.M Bahnson T.D Yu H Kim N.N Printz M.P Species variability in angiotensin receptor expression by cultured cardiac fibroblasts and the infarcted heart Am J Physiol 1998 274 H801 H809 Google Scholar PubMed OpenURL Placeholder Text WorldCat [15] Gillis K.D Sakmann B.N.E Techniques for membrane capacitance measurement Single-channel recording 2000 London Plenum Press 155 198 [16] Von Beckerath N Dittrich M Klieber H.G Daut J Inwardly rectifying K+ channels in freshly dissociated coronary endothelial cells from guinea-pig heart J Physiol (Lond) 1996 491 357 365 Google Scholar Crossref Search ADS PubMed WorldCat [17] Yokoyama T Sekiguchi K Tanaka T et al. Angiotensin II and mechanical stretch induce production of tumor necrosis factor in cardiac fibroblasts Am J Physiol 1999 276 H1968 H1976 Google Scholar PubMed OpenURL Placeholder Text WorldCat [18] Ganitkevich V.Y Isenberg G Stimulation-induced potentiation of T-type Ca2+ channel currents in myocytes from guinea-pig coronary artery J Physiol (Lond) 1991 443 703 725 Google Scholar Crossref Search ADS PubMed WorldCat [19] Nilius B Eggermont J Voets T Droogmans G Volume-activated Cl-channels Gen Pharmacol 1996 27 1131 1140 Google Scholar Crossref Search ADS PubMed WorldCat [20] Kamkin A Kiseleva I Isenberg G Stretch-activated currents in ventricular myocytes: amplitude and arrhythmogenic effects increase with hypertrophy Cardiovasc Res 2000 48 409 420 Google Scholar Crossref Search ADS PubMed WorldCat [21] Kamkin A Kiseleva I Wagner K.D et al. Mechanically induced potentials in fibroblasts from human right atrium Exp Physiol 1999 84 347 356 Google Scholar Crossref Search ADS PubMed WorldCat [22] Kamkin A Kiseleva I Wagner K.D et al. Mechanically induced potentials in rat atrial fibroblasts depend on actin and tubulin polymerization Eur J Physiol 2001 442 487 497 Google Scholar Crossref Search ADS WorldCat [23] Yang X.C Sachs F Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions Science 1989 243 1068 1071 Google Scholar Crossref Search ADS PubMed WorldCat [24] Hongo K Pascarel C Cazorla O et al. Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular myocytes Exp Physiol 1997 82 647 656 Google Scholar Crossref Search ADS PubMed WorldCat [25] Brilla C.G Scheer C Rupp H Angiotensin II and intracellular calcium of adult cardiac fibroblasts J Mol Cell Cardiol 1998 30 1237 1246 Google Scholar Crossref Search ADS PubMed WorldCat [26] Stockbridge L.L French A.S Characterization of a calcium-activated potassium channel in human fibroblasts Can J Physiol Pharmacol 1989 67 1300 1307 Google Scholar Crossref Search ADS PubMed WorldCat [27] Sachs F Morris C.E Mechanosensitive ion channels in nonspecialized cells Rev Physiol Biochem Pharmacol 1998 132 1 77 Google Scholar PubMed OpenURL Placeholder Text WorldCat [28] Zhang Y Gao F Popov V.L Wen J.W Hamill O.P Mechanically gated channel activity in cytoskeleton-deficient plasma membrane blebs and vesicles from Xenopus oocytes J Physiol (Lond) 2000 523 117 130 Google Scholar Crossref Search ADS PubMed WorldCat [29] Crenshaw H.C Allen J.A Skeen V Harris A Salmon E.D Hydrostatic pressure has different effects on the assembly of tubulin, actin, myosin II, vinculin, talin, vimentin, and cytokeratin in mammalian tissue cells Exp Cell Res 1996 227 285 297 Google Scholar Crossref Search ADS PubMed WorldCat Copyright © 2003, European Society of Cardiology
TI - Activation and inactivation of a non-selective cation conductance by local mechanical deformation of acutely isolated cardiac fibroblasts
JF - Cardiovascular Research
DO - 10.1016/S0008-6363(02)00775-7
DA - 2003-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/activation-and-inactivation-of-a-non-selective-cation-conductance-by-lAkIltkzjY
SP - 793
EP - 803
VL - 57
IS - 3
DP - DeepDyve
ER -