Andre Kamkin

Electrical interaction of mechanosensitive fibroblasts and myocytes in the heart

Fibroblasts in the heart can respond to mechanical deformation of the plasma membrane with characteristic changes of their membrane potential. Membrane depolarization of the fibroblasts occurs during the myocardial contractions and is caused by an influx of cations, mainly of sodium ions, into the cells. Conversely, application of mechanical stretch to the cells, i.e., during diastolic relaxation of the myocardium, will hyperpolarize the membrane potential of the fibroblasts due to reduced sodium entry. Thus, cardiac fibroblasts can function as mechano–electric transducers that are possibly involved in the mechano–electric feedback mechanism of the heart. Mechano–electric feedback refers to the phenomenon, that the cardiac mechanical environment, which depends on the variable filling pressure of the ventricles, modulates the electrical function of the heart. Increased sensitivity of the cardiac fibroblasts to mechanical forces may contribute to the electrical instability and arrhythmic disposition of the heart after myocardial infarction. Novel findings indicate that these processes involve the intercellular transfer of electrical signals between fibroblasts and cardiomyocytes via gap junctions. In this article we will discuss the recent progress in the electrophysiology of cardiac fibroblasts. The main focus will be on the intercellular pathways through which fibroblasts and cardiomyocytes communicate with each other.

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.

Role of nitric oxide in activity control of mechanically gated ionic channels in cardiomyocytes: NO-donor study.

Whole-cell ionic currents through mechanically gated chnnels (MGC) were recorded in isolated cardiomyocytes under voltage clamp conditions. In unstrained cells, NO donors SNAP and DEA-NO activated MGC and induced MG-like currents. In contrast, in stretched cells with activated MGC, these NO-donors inactivated and inhibited MGC.

Role of Nitric Oxide in the Regulation of Mechanosensitive Ionic Channels in Cardiomyocytes: Contribution of NO-Synthases

The role of NO in the regulation of currents passing through ion channels activated by cell stretching (mechanically gated channels, MGC), particularly through cation-selective K+-channels TRPC6, TREK1 (K2P2.1), and TREK2 (K2P10.1), was studied on isolated mouse, rat, and guinea pig cardiomyocytes using whole-cell patch-clamp technique. In non-deformed cells, binding of endogenous NO with PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline- 1-1-oxy-3-oxide) irreversibly shifted the diastolic membrane potential towards negative values, modulates Kir-channels by reducing IK1, and blocks MGC. Perfusion of stretched cells with PTIO solution completely blocked MG-currents. NO-synthase inhibitors L-NAME and L-NMMA completely blocked MGC. Stretching of cardiomyocytes isolated from wild type mice and from NOS1–/–- and NOS2–/–- knockout mice led to the appearance in MG-currents typical for the specified magnitude of stretching, while stretching of cardiomyocytes from NOS3–/–- knockout mice did not produce in MG-current. These findings suggest that NO plays a role in the regulation of MGC activity and that endothelial NO-synthase predominates as NO source in cardiomyocyte response to stretching.

The Role of Nitric Oxide in the Regulation of Mechanically Gated Channels in the Heart

The article presents the effects of NO on myocardial functions including its pronounced influence on myocardium contraction and heart rhythm. Attention is given to cell signaling of nitric oxide in the heart. It is demonstrated that in general the final effect of NO depends on the cellular source of NO, amount of NO release, the prevailing redox balance and antioxidant status, stimuli such as coronary flow rate and heart rate, the target tissue, interaction with neurohumoral and other stimuli, activity level of the immune system and activation of cGMP-dependent and independent intracellular cascades. A number of experiments conducted on whole hearts lets us suppose that NO and NO-synthases as NO origins, directly regulate the conductivity of mechanically gated channels (MGCs). This study discusses experimental data obtained from isolated ventricular myocytes of mouse, rat and guinea pig by means of patch-clamp in the whole-cell configuration about the role of NO in the regulation of MGCs. Presented data demonstrate that NO donors lead to MGCs activation and appearance of MG-like currents in unstretched ventricular myocytes, while in stretched cells with activated MGCs NO donors lead to inactivation and inhibition of the conductivity of these channels. The NO scavenger PTIO causes inactivation of all MGCs. In unstretched cells the conductance through MGCs is blocked, which is present in control before deformation. PTIO causes complete inhibition of stretch induced MG-current during presence of cellular stretch. Application of non selective inhibitors of NO-synthases L-NAME or L-NMMA resulted in a complete blockade of MGCs. The presented data are instituted on cells of transgenic mice. In ventricular myocytes of wild-type mice, NOS1–/– and NOS2–/– stretching of cells results in an activation of typical MG-currents. On the contrary, in cells from NOS3–/– mice stretch does not activate MG-currents. The results suggest that NO plays an important role in the activation and inactivation of MGCs in cardiomyocytes and demonstrate that NOS3 dominates as NO origin.

Mechanosensitive Alterations of Action Potentials and Membrane Currents in Healthy and Diseased Cardiomyocytes: Cardiac Tissue and Isolated Cell

Several electrophysiological alterations in the heart, which were ascribed to mechanoelectric feedback have been reported. First of all, they include changes in mechano-gated channels, mechanosensitive whole-cell currents which lead to membrane depolarization which is equivalent to a decrease in the resting membrane potential and elicited stretch-induced depolarizations, that appear during repolarization phase of cardiomyocyte action potential. Stretch-induced depolarization during action potentials provoke extra-action potentials when the stretch-induced depolarizations reach a threshold potential. Mechano-gated channels and mechanosensitive whole-cell currents are the cellular meachanisms underlying this phenomenon. In this review we discuss some open questions about mechanosensitive ionic currents in freshly isolated single cardiomyocytes. We will demonstrate certain methods of direct mechanical deformation of isolated cardiomyocytes for the purpose of electrophysiological investigation, including different experimental approaches to application of stretch and compression to pressure the cardiomyocytes. It is necessary to note that brick-like isolated cardiomyocytes stick to the bottom of the perfusion chamber in two different positions: edgewise, staying on the narrow side, or broad-wise. Partly these different positions of cells define the cell reaction to deformation. The reaction to stretch is identical in cardiomyocytes, occupying both positions (edgewise and broad-wise). However, the reaction to compression is different and is determined by the position of a cell. We demonstrate the possibility of simultaneous recording of mechano-gated single channels (in cell-attached mode) and mechanosensitive whole-cell currents during direct deformation of the whole cell. We discuss the results of stretch and compression of freshly isolated atrial cardiomyocytes from healthy and diseased animals and humans. Isolated cardiomyocytes respond to stretch with membrane depolarization, prolongation of their action potential (AP) and extra-APs that correlated with the amplitude of a non-selective stretch-activated current (ISAC). At negative potentials, ISAC is negative and carried by a transmembrane influx of Na+ ions. In this review we discuss some of the recent advances from intracellular recordings of the bioelectrical activity of cardiomyocytes during mechanical stretch of healthy and diseased tissues from animals and humans. The sensitivity of the AP to mechanical stretch was significantly increased in hypertrophied myocardium, and this could be related to the expression of SACs. We suppose that they are the basic findings that may explain mechanism of some arrhythmias and fibrillation.

The Role of Mechanosensitive Fibroblasts in the Heart: Evidence from Acutely Isolated Single Cells, Cultured Cells and from Intracellular Microelectrode Recordings on Multicellular Preparations from Healthy and Diseased Cardiac Tissue

Cardiac fibroblasts are electrically non-excitable cells that respond to mechanical deformation of the cells with typical changes of their membrane potential. These changes of fibroblasts membrane potential are determined by operation of mechano-gated channel (MGCs). Two types of MGCs with conductances of 43 pS and 87 pS were observed during direct deformation of the fresh isolated cells. Cell compression augment the whole-cell MG currents and increase the frequency and duration of single MGC openings. Both MGCs (with conductance levels of 43 pS and 87 pS) displayed linear current-voltage relationships with the reversal potential around 0 mv. Cell stretch inactivated the whole-cell MG currents and abolished the activity of single MGCs. These channels, mainly permeable for sodium ions, are activated by compression of the cell leading to depolarization, and are inactivated by stretch, which in turn leads to hyperpolarization. Cultured cardiac fibroblasts preserve MGCs up to 5 days without special flexible substrates and possess electrophysiological properties of freshly isolated cells. Thus, cardiac fibroblasts function as mechano-electric transducers in the heart and represent the cellular substrate for a cardiac mechano-electrical feedback mechanism. Cardiac fibroblasts 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 is thought to participate in the mechano-electric feedback mechanism of the heart. Enhanced sensitivity of the cardiac fibroblasts to mechanical deformation is increasing with age and during hypertrophy. It is contributing to electrical instability and arrhythmia after myocardial infarction. Recent findings indicate that these processes involve the transfer of electrical signals via gap junctions. In this article we will discuss recent progress in the electrophysiology of cardiac fibroblasts and their role in mechano-electric feedback in healthy and diseased hearts.

Mechanical Stretching of Cells of Different Tissues: The Role of Mediators of Innate Immunity

The current review describes the modern conce of how the mechanical stretch (MS) affects cytokine and chemokine production by the cells of different tissues (cardiomyocytes, fibroblasts, smooth muscle cells, endothelial cells and pulmonary cells). Released mediators regulate cell functions such as synthesis of the extracellular matrix proteins, proliferation, apoptosis and others, in autocrine or paracrine manner. Endogenous cytokines (tumor necrosis factor α (TNFα), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), interleukin 6 (IL-6) and others) produced in myocardium in response to mechanical stretch (MS) may trigger pathological processes resulting in myocyte growth, apotosis and formation of reactive fibrosis. Mechanical load is associated with increase in tissue volume and tissue remodeling. This review provides data about changes in expression of cytokine receptors expression, as well as receptors of innate immunity (TLRs), in response to MS. TLR4 is expressed on the surface of cells of the heart, including cardiomyocytes, smooth muscle cells and endothelial cells. Cyclic MS enhances expression of TLR4 in cultured neonatal rat cardiomyocytes. Excessive MS may result in alterations of cell structure and functions, composition of extracellular matrix (ECM), and promote development of pathological conditions such as hypertrophy, fibrosis, atherosclerosis, osteoporosis, etc. Searching for drugs with targeted action working at the extracellular, membrane and intracellular levels and which will improve the consequences of excessive MS is of undoubted interest and is actual for the treatment of many human pathologies.

Experimental Methods of Studying Mechanosensitive Channels andPossible Errors in Data Interpretation

In this review we discuss most widely used experimental methods of the membrane stretch which are used for investigation of mechanosensitive channels (MSCs) by patch-clamp. We have tried to discuss possible mistakes in interpreting the data received by various methods of MSCs investigation. In the conditions of single channel recording we briefly analyse positive and negative pressure as mechanical stimulation and demonstrate that MSC respond only to membrane tension. After gigaseal forming suction there appears resting patch for the reason of the patch adhesion to the glass and this creates a resting tension. It is shown that some channels can be active at zero pressure because the seal adhesion energy produces tension. Such a situation can be considered as pre-stretch. Related to this we discuss research showing that stretch-inactivated channels (SICs) do not imply the existence of a new type of channel, but inactivation of channel activity in response to suction can be explained by the activity of pre-stressing of stretch-activated channels (SACs). We also criticize the presence of pressure activated channels (PACs). According to the Laplace’s equation, positive or negative pressures should make equal contributions to the stress. In the conditions of whole cell recording we discuss the known methods of a cell direct mechanical stretching. That is homogeneous stretching of single cells with the use of two patch pipettes, three types of axial stretch - by two glass capillaries, by glass stylus and by two thin carbon fibres. We briefly discuss the merits and imperfections of cell swelling. We analyse the possibilities of paramagnetic microbead method that allows the application of controlled forces to the membrane at which those mechanical forces are transmitted by integrins. We discuss the possibilities of cell compression. Obviously the stresses are very complicated in compression and no one knows how to analyze the data in a mechanistic manner. We discuss the study of bacterial mechanosensitive channels. We discuss the limitation of the research using protein purification and functional reconstitution in planar lipid bilayers or in vesicles. Also, rarely used methods are presented. In this review we discuss most widely used experimental methods of the membrane stretch, which are used for investigation of mechanosensitive channels (MSCs) by means of patch-clamp method. We address possible mistakes in interpreting the data, obtained by means of various methods of MSCs investigation. Under conditions of single channel recording we briefly analyse positive and negative pressure in terms of mechanical stimulation and demonstrate that MSC respond only to membrane tension. Resting tension of the membrane is created after suction, which is applied for the purpose of gigaseal formation. It is shown that some channels can be active at zero pressure because the seal adhesion energy produces tension. Such situation can be considered as pre-stretch. In this respect we discuss reports, showing that stretch-inactivated channels (SICs) do not imply the existence of a new type of channels, when inactivation of channel activity in response to suction can be explained by the activity of pre-stressing of stretch-activated channels (SACs). We discuss the controversy about the presence of pressure activated channels (PACs). According to the Laplace’s equation, positive or negative pressures should make equal contributions to the stress. We also discuss reported methods of direct mechanical stretching of cells during whole cell recording. Discussion covers method of homogeneous stretching of a single cell by means of two patch pipettes and three types of axial stretch - by two glass capillaries, by glass stylus and by two thin carbon fibres. We briefly discuss the merits and imperfections of cell swelling. We analyse the possibilities of paramagnetic microbead method that allows the application of controlled forces to the membrane, at the level of which those mechanical forces are transmitted by integrins. We discuss possible methods of cell compression. Obviously distribution of forces is very complicated during compression and no one knows how to analyze the data in a mechanistic manner. We discuss the study of bacterial mechanosensitive channels. We discuss the limitation of the research using protein purification and functional reconstitution in planar lipid bilayers and in vesicles. Also, rarely used methods are presented

The Role of Nitric Oxide in the Regulation of Ion Channels in the Cardiomyocytes: Link to Mechanically Gated Channels

This review is devoted to the role of nitric oxide in the regulation of ion channels in the cardiomyocytes. Here we consider issues of regulation of mechanically gated channels by means of NO. Firstly we address the modulatory effect of nitric oxide on voltage gated Na+-, Ca2+-, K+-channels, which contribute them most to formation of action potential and its shape under normal as well as under pathological conditions in heart. We address separately effect of nitric oxide on leak channels (two-pore potassium channels), some of which are mechanosensitive. Finally we discuss the effect of nitric oxide on mechanically gated ion channels and mechanically gated currents. In our manuscript we show that without consideration of NO effects on voltage gated channels, investigation of nitric oxide effect on mechanically gated channels in heart under normal of pathological conditions would be incomplete.