Kamkin.doc

CONTRIBUTION OF FIBROBLASTS TO MECHANOELECTRIC
FEEDBACK IN HEALTHY AND DISEASED HEARTS
Andre Kamkin1, Irina Kiseleva1, Kay-Dietrich Wagner2, Joachim Günther2,
Holger Scholz2 and Gerrit Isenberg3
1Department of Fundamental and Applied Physiology, Russian States Medical University, Ostrovitjanova 1, 117997 Moscow, Russia. 2Institut für Physiologie, Charité, Humboldt - Universität, Tucholskystrasse 2, 10117 Berlin Germany. 3Institut für Physiologie, Martin-Luther-Universität Halle-Wittenberg, Magdeburger Str. 6, 06097 Halle/Saale, Germany. Electrophysiological studies of cardiac fibroblasts started with the right atrium of the frog heart [1]. Electrophoretically injected Lucifer Yellow or colloidal gold demonstrated that the microelectrode recordings were indeed from fibroblasts. The fibroblast resting membrane potentials (Em) varied between – 70 mV and – 5 mV, on average Em was -31±3 mV in frog [1], -22±2 mV in rat, [2], and 16±2 mV in human atria [3]. The fibroblast input resistance (Rin) was 1.1±0.1 GΩ (frog, [1]), 510±10 MΩ (rat [2]), 4.4±0.1 GΩ (human atria, [3]). Cardiac fibroblasts are non-excitable. They respond with mechanically induced potentials (MIPs) to mechanical stimuli such as the spontaneous contractions of the surrounding myocardium or short-time mechanical stretches of the multicellular strip during an experiment. MIPs depolarized Em towards a reversal potential of approx. –5 mV and were reversibly reduced by Gd3+ (40 µM), both properties suggesting that the MIP-depolarisation could be caused by a non-selective cation conductance Gns that is activated by the mechanical deformation of the fibroblast surface by the contracting atrial myocytes, in analogy to the stretch-activated channels (SACs). When atrial strips were stretched by a micrometer screw, the fibroblasts hyperpolarized and the amplitude of MIPs (AMIP) increased. Polarization of Em by current injection changed AMIP but not the frequency of MIPs. AMIP was 38±4 mV when Em was set to Vm=-50 mV. The slope of the function relating AMIP to Vm described the relative increase in Gns during a MIP (‘mechanosensitive factor’ Xms). The involvement of the cytoskeleton in activation of Gns was studied by delivering drugs into the fibroblast from the intracellular recording microelectrode. Destabilisation of F-actin by 0.2 mM cytochalasin D reduced AMIP from 38 to 16 mV and Xms from 5 to 1.8. Destabilisation of tubulin with 0.2 mM colchicine reduced AMIP to 21 mV and Xms to 2.1. The combination of colchicine plus cytochalasin D reduced AMIP to 9 mV and Xms to 1.4. Promoting F-actin stability with exogenous ATP increased AMIP and Xms and attenuated the effects of cytochalasin D. Similarly, facilitation of tubulin stability with GTP or taxol increased AMIP and Xms and attenuated the effects of colchicine. The results suggest that the cytoskeleton is involved in the transfer of mechanical energy from the deformed fibroblast surface to the SAC protein [4]. The hypothesis whether mechanically induced Ca2+ influx through SACs and/ or release of Ca2+ from the endoplasmic reticulum are involved in the generation of MIP [5] was studied by releasing BAPTA, BHQ, thapsigargin, CPA or ryanodine from the microelectrode into the fibroblast. All the compounds decreased AMIP to variable degrees. MIP duration was reduced by most interventions, exceptions being low extracellular Ca2+, BHQ and ryanodine. A short extracellular application of caffeine partly restored the MIP by activation of cardiac contraction. Intracellular current injection altered both, Vm and AMIP in a linear way before, however, non-linearly after the above compounds had been applied. The results were thought to support the hypothesis that changes in intracellular Ca2+ concentration play a role in the generation of MIPs. Isolated rat atrial fibroblasts were used for a voltage-clamp analysis of the ionic currents generating MIPs. The fibroblast was mechanically deformed between two patch-pipettes, a first pipette was used for whole cell clamp and served as a fix point. A second cell attached pipette was laterally displaced in regard to first pipette, thereby compressing or stretching the cell. The dependence of ionic current on membrane potential (I-V relation) was measured by applying 20 pulses of 140 ms (0.5 Hz) that started from a holding potential of –45 mV. When the membrane currents flowing at the end of the pulse (IL) were plotted versus the respective clamp step potential, the resulting I-V curve intersected the voltage axis at the zero current potential (E0, corresponding to the resting potential of non-clamped cells). In isolated fibroblasts, E0 varied between -70 and -10 mV. On average, the isolated rat atrial fibroblast had an E0 (-37±3 mV, n=50) and an input resistance Rin (514±11 MΩ) that resembled their counterparts in multicellular rat atrial strips. Compression by 2, 3, or 4 µm shifted E0 from -34±5 to -24±3 mV (n=4), to -10±3 mV (n=4), and to -5±2 mV (n=5), respectively. The stretch sensitive difference followed a modest outward rectification, the amplitude (at –45 mV) of which increased with the extent of compression (-0.03±0.01 at 2 µm, -0.18±0.05 at 3 µm, and -0.34±0.09 nA at 4 µm) whilst the reversal potential was nearly constant (Erev +5±3, +1±2, +6±3). The currents were carried by Na+, K+ and Cs+ ions, were blocked by extracellular application of 8 µM Gd3+. The results suggest that compression activates a non-selective cation conductance Gns. Stretch by 2, 3, or 4 µm hyperpolarized E0 from -35±6 to-43±6 (n=4), to -52±3 (n=4) or to -82±8 mV (n=4). Stretch induced a positive difference current (at –45 mV: 0.05±0.02, 0.12±0.04, and 0.37±0.03 nA) with a Erev close to 0 mV (0±1, -6±1, and -10±2 mV, respectively). Application of Gd3+ during continuous stretch shifted E0 to potentials as negative as EK (-97±5 mV). Ion selectivity, Erev and Gd3+ sensitivity of stretch suppressed currents let us suggest that stretch reduces the non-selective cation conductance Gns that is activated by compression. Cell dialysis with 5 mM BAPTA (pCa >8) or 5 mM Ca2+/EGTA (pCa =6) had no influence on the compression-activated or stretch-deactivated currents suggesting that Ca2+ dependent conductances are unlikely to contribute. The results could be modelled on the assumption of a compression-activated, stretch-deactivated Gns operating in parallel to a mechano-insensitive K+ conductance and a current due to electrogenic Na+ pumping. During remodelling after myocardial infarction (MI), both number and electrical properties of atrial fibroblasts change. We tested whether changes in fibroblast membrane potential were related to the infarct size (IS), the degree of hypertrophy, and the time after MI. After the small, intermediate and large IS, the resting potential was -25.8±1.9, -35.9±1.6, -46.5±1.8 mV, respectively. At 20 days after MI, the degree of atrial hypertrophy (cell number) correlated positively with IS. The sensitivity of the fibroblast membrane potential to stretch increased in proportion to IS. Time-dependent changes of rat atrial fibroblast membrane potentials were studied at constant infarct size of 16%. Whilst ventricular hypertrophy increased between 8 and 30 days after infarction, the sensitivity of atrial fibroblast membrane potential to stretch fell. In vivo heart rates and the rates of spontaneous atrial contractions were significantly reduced on day 8 after infarction indicating bradycardia, and recovered in parallel to the membrane potential of the atrial fibroblasts. We discuss that this altered atrial fibroblast electrophysiology may contribute to the bradycardia during post-infarct remodelling on the assumption that in the sino-atrial node region cardiac fibroblasts electrically couple to the surrounding cardiomyocytes [2]. 1. Kiseleva IS, Kamkin AG, Kircheis R, Kositzky GI. Intracellular electrotonical interaction in sinus region of the frog heart. Reports of Acad of Sci of Soviet Union. 1987; 292 (6): 1502-1505.
2. Kohl P, Kamkin AG, Kiseleva IS, Noble D. Mechanosensitive fibroblasts in the sino-atrial node region of heart: interaction with cardiomyocytes and possible role. Exp Physiol. 1994; 79: 943-
956.
3. Kamkin A, Kiseleva I, Wagner KD, Lammerich A, Bohm J, Persson PB, Günther J. Mechanically induced potentials in fibroblasts from human right atrium. Exp Physiol. 1999; 84:
347-356.
4. Kamkin A, Kiseleva I, Wagner KD, Scholz H, Theres H, Kazanski V, Lozinsky I, Günther J, Isenberg G. Mechanically induced potentials in rat atrial fibroblasts depend on actin and tubulin
polymerization. Eur J Physiol. 2001; 442: 487-497.
5. Kiseleva I, Kamkin A, Kohl P, Lab MJ. Calcium and mechanically induced potentials in fibroblasts of rat atrium. Cardiovasc Res. 1996; 32: 98-111.

Source: http://mef.physiol.ox.ac.uk/docs/lectures/kamkin.pdf