Irina Kiseleva

Mechanosensitive fibroblasts in the sino‐atrial node region of rat heart: interaction with cardiomyocytes and possible role

The positive chronotropic response of the heart to stretch of the right atrium is one of the major mechanisms adjusting the heart rate to variations in venous return on a beat‐by‐beat basis. The precise pathway of this mechano‐electric feedback and its cellular basis are uncertain. In this study, a possible contribution of mechanosensitive fibroblasts, abundant in the sino‐atrial node region, was investigated using a mathematical model of the electrical interaction of a mechanosensitive fibroblast and a sino‐atrial pacemaker cell. Electrophysiological evidence for a bio‐electrical interaction of mechanosensitive fibroblasts with surrounding cardiomyocytes has been studied in (i) the isolated spontaneously beating atrium of rat hearts, and (ii) cell cultures of the neonatal rat heart. These investigations were performed using (i) double‐barrelled floating microelectrodes for intracellular potential registrations, and (ii) the double whole cell patch‐clamp technique. It was shown that cardiac fibroblasts and surrounding cardiomyocytes can be either electrically well isolated from each other, or coupled both capacitively and electrotonically. The electrophysiological data obtained were incorporated into the OXSOFT HEART program. Assuming that equivalent coupling may occur between mechanosensitive fibroblasts and sino‐atrial pacemaker cells, a heterologous cell pair consisting of one fibroblast and one sino‐atrial node myocyte connected by ten to thirty single gap junctional channels with a conductance of 30 pS was modelled. The model of the electrotonic interaction of these cells showed that stretch of the fibroblast during atrial diastole, simulating increased atrial wall tension during atrial filling, can raise the spontaneous depolarization rate of the pacemaker cell in a stretch‐dependent manner by up to 24%. These results show that cardiac mechanosensitive fibroblasts could form a cellular basis for the positive chronotropic response of the heart to stretch of the right atrium.

Inotropic response to β-adrenergic receptor stimulation and anti-adrenergic effect of ACh in endothelial NO synthase-deficient mouse hearts

1 The functional consequences of a lack of endothelial nitric oxide synthase (eNOS) on left ventricular force development and the anti‐adrenergic effect of acetylcholine (ACh) were investigated in isolated hearts and cardiomyocytes from wild type (WT) and eNOS knockout (eNOS–/–) mice. 2 eNOS expression in cardiac myocytes accounted for 20 % of total cardiac eNOS (Western blot analysis). These results were confirmed by RT‐PCR analysis. 3 In the unstimulated perfused heart, the left ventricular pressure (LVP) and maximal rate of left ventricular force development (dP/dtmax) of eNOS–/– hearts were not significantly different from those of WT hearts (LVP: 97 ± 11 mmHg WT vs. 111 ± 11 mmHg eNOS–/–; dP/dtmax: 3700 ± 712 mmHg s−1 WT vs. 4493 ± 320 mmHg s−1 eNOS–/–). 4 The dobutamine (10‐300 nm)‐induced increase in LVP was enhanced in eNOS–/– hearts. In contrast, L‐type Ca2+ currents (ICa,L) in isolated cardiomyocytes of WT and eNOS–/– hearts showed no differences after β‐adrenergic stimulation. Dibutyryl‐cGMP (50 μm) reduced basal ICa,L in WT cells to 72 ± 12 % while eNOS–/–ICa,L was insensitive to the drug. The pre‐stimulated ICa,L (30 nm isoproterenol) was attenuated by dibutyryl‐cGMP in WT and eNOS–/– cells to the same extent. 5 The Ca2+ (1.5‐4.5 mm)‐induced increase in inotropy was not different between the two experimental groups and β‐adrenergic receptor density was increased by 50 % in eNOS–/– hearts. 6 The contractile effects of dobutamine could be inhibited almost completely by ACh or adenosine. The extent of the anti‐adrenergic effect of both compounds was identical in WT and eNOS–/– hearts. Measurement of ICa,L in isolated cardiac myocytes yielded similar results. 7 These data demonstrate that in the adult mouse (1) lack of eNOS is associated with increased cardiac contractile force in response to β‐adrenergic stimulation and with elevated β‐adrenergic receptor density, (2) the unaltered response of ICa,L in eNOS–/– cardiac myocytes to β‐adrenergic stimulation suggests that endothelium‐derived NO is important in mediating the whole‐organ effects and (3) eNOS is unimportant for the anti‐adrenergic effect of ACh and adenosine.

Differential effects of stretch and compression on membrane currents and [Na+]c in ventricular myocytes.

Mechano-electrical feedback was studied in the single ventricular myocytes. A small fraction (approximately 10%) of the cell surface could be stretched or compressed by a glass stylus. Stretch depolarised, shortened the action potential and induced extra systoles. Stretch activated non-selective cation currents (I(ns)) showed a linear voltage dependence, a reversal potential of 0 mV, a pure cation selectivity, and were blocked by 8 microM Gd(3+) or 30 microM streptomycin. Stretch reduced Ca(2+) and K(+) (I(K)) currents. Local compression of broadwise attached cells activated I(K) but not I(ns). Cytochalasin D or colchicin, thought to disrupt the cytoskeleton, suppressed the mechanosensitivity of I(ns) and I(K). During stretch, the cytosolic sodium concentration increased with spatial heterogeneities, local hotspots with [Na(+)](c)>24 mM appeared close to surface membrane and t-tubules (pseudoratiometric imaging using Sodium Green fluorescence). Electronprobe microanalysis confirmed this result and indicated that stretch increased total sodium [Na] in cell compartments such as mitochondria, nuclear envelope and nucleus. Our results obtained by local stretch differ from those obtained by end-to-end stretch (literature). We speculate that channels may be activated not only by axial but also by shear stress, and, that stretch can activate channels outside the deformed sarcomeres via second messenger.

Activation and inactivation of a non-selective cation conductance by local mechanical deformation of acutely isolated cardiac fibroblasts

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 (E(0)) of -37+/-3 mV and an input resistance of 514+/-11 M(Omega). At intracellular pCa 7 (patch-pipette solution), compression of 2 or 3 microm shifted E(0) from -36+/-7 to -17+/-3 mV, and to -10+/-2 mV. Compression by 2 or 3 microm induced a negative difference current (at -45 mV -0.06+/-0.02 and -0.20+/-0.04 nA, respectively) with a reversal potential (E(rev)) of approx. 0 mV. The currents were carried by Na(+), K(+) and Cs(+) ions, and were blocked by application of 8 microM Gd(3+). Stretch of 2 or 3 microm hyperpolarized E(0) 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 E(rev) close to 0 mV. Application of Gd(3+) shifted E(0) to potentials as negative as E(K) (-90+/-4 mV). Cell dialysis with 5 mM BAPTA (pCa 8) or 5 mM Ca(2+)/EGTA (pCa 6) had no influence on non-selective cation currents suggesting that Ca(2+) 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 (G(ns)). Stretch hyperpolarizes the fibroblast, however, not by Ca(2+) activation of K(+)-conductance. Ion selectivity, E(rev,) and Gd(3+)-sensitivity of stretch suppressed currents suggest that stretch reduces G(ns) that is activated by compression.

Cardiac fibroblasts and the mechano-electric feedback mechanism in healthy and diseased hearts.

Cardiac arrhythmia is a serious clinical condition, which is frequently associated with abnormalities of mechanical loading and changes in wall tension of the heart. Recent novel findings suggest that fibroblasts may function as mechano-electric transducers in healthy and diseased hearts. Cardiac fibroblasts are electrically non-excitable cells that respond to spontaneous contractions of the myocardium with rhythmical changes of their resting membrane potential. This phenomenon is referred to as mechanically induced potential (MIP) and has been implicated in the mechano-electric feedback mechanism of the heart. Mechano-electric feedback is thought to adjust the frequency of spontaneous myocardial contractions to changes in wall tension, which may result from variable filling pressure. Electrophysiological recordings of single atrial fibroblasts indicate that mechanical compression of the cells may activate a non-selective cation conductance leading to depolarisation of the membrane potential. Reduced amplitudes of MIPs due to pharmacological disruption of F-actin and tubulin suggest a role for the cytoskeleton in the mechano-electric signal transduction process. Enhanced sensitivity of the membrane potential of the fibroblasts to mechanical stretch after myocardial infarction correlates with depression of heart rates. It is assumed that altered electrical function of cardiac fibroblasts may contribute to the increased risk of post-infarct arrhythmia.

Mechanically Induced Potentials in Fibroblasts from Human Right Atrium

It has been shown that cardiac fibroblasts of the human heart are electrically non‐excitable and mechanosensitive. The resting membrane potential of these cells is ‐15.9 ± 2.1 mV and the membrane resistance is 4.1 ± 0.1 GΩ. Rhythmic contractions of the myocardium associated with stretch of the surrounding tissue produce reversible changes in the membrane potential of cardiac fibroblasts. These mechanically induced potentials (MIPs) follow the rhythm of myocardial contractions. Simultaneous recording of the action potential of cardiomyocytes and MIPs of cardiac fibroblasts demonstrates a delay of 40.0 ± 0.4 ms after the action potential before the appearance of the MIP. Contraction produces a MIP which is more positive or more negative than the reversal potential ‐ the membrane potential due to current injection at which the MIP reverses its direction. Regardless of the initial orientation of the MIP, intracellular polarization increases the amplitude towards the reversal potential if the background MIP had depolarized the membrane or away from the reversal potential if the initial background MIP had hyperpolarized the membrane. Artificial intracellular polarization changed the amplitude but not the frequency of the MIP. The pool of electrically non‐excitable mechanosensitive cells, which change their electrical activity during contraction and relaxation of the heart, may play a role in the mechano‐electrical feedback mechanism which has to be taken into account in the normal function of the heart as well as in pathological processes.

Calcium and mechanically induced potentials in fibroblasts of rat atrium

OBJECTIVES Electrically non-excitable cardiac fibroblasts in the sino-atrial node region are mechano-sensitive. Rhythmic contraction of adjacent myocardium, or artificial stretch of the tissue, produce a reversible change in the membrane potential: mechanically induced potentials (MIP). Stretch of normal cardiomyocytes can be associated with intracellular calcium changes. The purpose of this study is to use pharmacological interventions to investigate the possibility that stretch-induced Ca2+ entry through ion channels in the sarcolemma and Ca2+ release from internal stores play a role in MIP generation. METHODS Isolated spontaneously contracting or artificially stretched preparations of right atrium of rat heart were superfused with physiological solutions. An intracellular floating microelectrode recorded fibroblast MIPs and was also used for injection of current. A dye, Lucifer yellow, applied through the micropipette, identified recording sites. We assessed the role of extracellular Ca2+ using EGTA in the bathing solution. For the role of intracellular Ca2+ in the generation of MIP, several substances that influence [Ca2+]i handling were applied intracellularly by diffusion from the recording microelectrode. These include: BAPTA (to chelate intracellular Ca2+); BHQ, thapsigargin and CPA (to deplete Ca2+ from intracellular stores by inhibition of the endoplasmic reticulum (ER) ATP Ca2+ pump), and caffeine and ryanodine (to induce ER Ca2+ release). RESULTS All the pharmacological compounds which were introduced intracellulary, and EGTA applied extracellularly, decreased the amplitude of the MIP to variable degrees. Only thapsigargin induced a bi-phasic response with an initial increase in MIP amplitude, followed by a decrease. MIP duration was reduced by most interventions, exceptions being low extracellular Ca2+, BHQ and ryanodine. Short duration extracellular application of caffeine, which was added to the perfusate as a secondary contractile stimulus, partly restored the MIPs by activation of cardiac contraction. Intracellular current injection, before any intervention, linearly altered both membrane potential (Em) and MIP amplitude (Vm). Application of compounds listed above introduced non-linearity to the Em/Vm relationship. CONCLUSION We suggest that mechanically induced Ca2+ influx, induced through stretch-activated channels in the plasma membrane, and release of Ca2+ from the endoplasmic reticulum, play key roles in the mechanism of MIP generation. Further, our results demonstrate the existence of functional ryanodine/caffeine-sensitive Ca2+ stores in cardiac fibroblasts.

Mechanoelectric feedback after left ventricular infarction in rats

BACKGROUND Myocardial infarction can lead to electrical abnormalities and rhythm disturbances. However, there is limited data on the electrophysiological basis for these events. Since regional contraction abnormalities feature prominently in infarction, we investigated whether stretch of myocardium from the infarction borderzone can modulate the electrophysiological properties of cardiomyocytes via mechanoelectric feedback providing a mechanism for post-infarction arrhythmia. METHODS Five weeks after experimental myocardial infarction (MI) in rats due to ligation of the left coronary artery (n = 26) or after sham operation (SO, n = 16), action potentials (AP) were measured in left ventricular preparations from the infarction borderzone. Sustained stretch was applied via a micrometer. RESULTS Preparations from MI generated spontaneous electrical and contractile activity. Cardiomyocytes from MI had a comparable AP amplitude, a more negative resting membrane potential, and a prolonged AP duration (APD) when compared to SO. In SO, stretch of 150 microns increased the APD90. This was associated with stretch activated depolarizations near APD90 (SAD-90). In MI, significantly lower stretch, of only 20 microns, elicited SAD-90s, or SADs near APD50 (SAD-50). Stretch-induced events were suppressed by gadolinium, at a concentration (40 microM) normally used to inhibit stretch-activated channels. CONCLUSION After MI, SADs are generated in the infarction borderzone at lower degrees of stretch. Increased sensitivity of the membrane potential of cardiac myocytes to mechanical stimuli may contribute to the high risk of arrhythmia after infarction. These SADs may involve the opening of stretch-activated channels.

Mechanosensitive cells in the atrium of frog heart

Mechanosensitive cells were found in the sinus venosus and right atrium of the frog heart. Their intracellular membrane potentials were studied in spontaneously beating hearts and in artificially stretched preparations. Membrane resistance was indirectly proportional to the stretch applied. The electrophysiological data and distribution of these cells in the heart led to the conclusion that they are cardiac fibroblasts.

Mechanosensitivity and Mechanotransduction

Foreword by Holger Scholz Editorial: Basic principles of mechanosensing and mechanotransduction in cells, Andre Kamkin and Irina Kiseleva List of Contributors Part I The Role of Cytoskeleton in Mechanosensitivity and Mechanotransduction: 1. Integrin-mediated mechanotransduction in vascular smooth muscle cells, Kay-Pong Yip, Lavanya Balasubramanian, James S. K. Sham 2. The role of actin cytoskeleton in mechanosensation, Tianzhi Luo and Douglas N. Robinson 3. Effect of Cytoskeleton on the Mechanosensitivity of Genes in Osteoblasts, Qiang Fu, Changjing Wu, Yourui Li, Yiping Zhang 4. Involvement of the cytoskeletal elements in articular cartilage mechanotransduction, Emma J Blain Part II Molecular Mechanisms of Mechanotransduction and Ion Channels Modifiers : 5. The role of nitric oxide in regulation of mechanically gated channels in the heart, Victor Kazanski, Andre Kamkin, Ekaterina Makarenko, Natalia Lysenko, Natalia Lapina, and Irina Kiseleva 6. Role of signaling pathways in the myocardial response to biomechanical stress and in mechanotransduction in the heart, Danny Guo, Zamaneh Kassiri, and Gavin Y. Oudit 7. Atomistic molecular simulation of gating modifier venom peptides - two binding modes and effects of lipid structure, Kazuhisa Nishizawa Part III Mechanosensing and Mechanotransduction in Vascular Cells: 8. Cellular and molecular effects of mechanical stretch on vascular cells, Kou-Gi Shyu 9. Role of Proteoglycans in Vascular Mechanotransduction, Aaron B. Baker Part IV Mechanotransduction in the Lung: 10. Control of TRPV4 and its effect on the lung, James C. Parker and Mary I Townsley 11. The Role of Protein-protein Interactions in Mechanotransduction: Implications in Ventilator Induced Lung Injury, Matt Rubacha and Mingyao Liu Part V Mechanosensing and Mechanotransduction in Bone and Joint Tissues: 12. Cellular mechanisms of mechanotransduction in bone, Suzanne R.L. Young and Fredrick M. Pavalko 13. The mechanosensitivity of cells in joint tissues: Role in the pathogenesis of joint diseases, Christelle Sanchez, Marianne Mathy-Hartert, Yves Henrotin Part VI Mechanosensitivity of Sensor Systems: 14. Primary Cilia are Mechanosensory Organelles in Vestibular Tissues, Surya Nauli Part VII Mechanosensing and Mechanotransduction in Blood Cells: 15. Mechanosensitive K+ Channels in Mouse B Lymphocytes: PLC-mediated Release of TREK-2 from Inhibition by PIP2, Sung Joon Kim and Joo Hyun Nam Index.