Ilya Lozinsky

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.

The Role of Proinflammatory Cytokines in Regulation of Cardiac Bioelectrical Activity: Link to Mechanoelectrical Feedback

In this review we cover key issues, concerned with the effects of cytokines in cardiac tissue and cardiac cells. We discuss the effects of proinflammatory cytokines under non-pathological conditions and their mechanisms dependent and independent of nitric oxide. The role of proinflammatory cytokines is considered in acute myocardial infarction and in heart failure. We also describe proinflammatory cytokines as inductors of arrhythmia. We discuss ionic current alternation as possible mechanisms of cytokines action in heart. We consider TNF-α as a possible player in this signaling cascade. It was shown that TNF-α induced alternation of transmembrane action potentials. Influence of TNF-α on transient outward current (I to), I Kur, I Kr, I Ks, I K1 is also reported. We discuss the interplay between TNF-α and Ca2+ current, influence of TNF-α on SERCA. Then we consider influence of IL-1 on action potentials, I Na, I Ca, I K. We also address the role of IL-2, IL-6, and IL-11. Finally using TNF-α and IL-6 as an example we discuss the effects of cytokines on mechanoelectrical feedback. Perfusion of cardiac tissue with TNF-α containing solution leads to abnormalities in cardiac electrical activity, majorly to prolongation of APD90 and appearance of hump-like depolarization at APD90 level. After reaching E c hump-like depolarization transforms into extra-AP, leading to sustained arrhythmias. TNF-α activates NO cardiomyocyte synthases and the rise of intracellular NO levels opens MGCs, which leads to sodium entry into the cell, which depolarizes cellular membrane, shifting resting potential towards E C. We proposed and proved that TNF-α triggered arrhythmias can be mediated through activation of MGCs. Stretching of preparations removed TNF-α. Perfusion of preparation with IL-6 containing solution leads to fibrillation in response to low levels of stretch. IL-6 mechanisms of action are mediated by NO synthases in cardiomyocytes. The circulating levels of TNF-α and IL-6 were found to be significantly higher in patients suffering from atrial fibrillation. It suggests a positive feedback between inflammation and atrial fibrillation. Proinflammatory cytokines are believed to be markers of atrial fibrillation, and more over, the key element in this positive feedback system.

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.

Ion Channels in Cardiac Fibroblasts: Link to Mechanically Gated Channels and their Regulation

Cardiac fibroblasts according to a number of authors have delayed rectifier current (IK), transient potassium current (Ito), inward rectifier potassium current (IKir), Ca2+-activated K+ current (IK(Ca)), TTX-sensitive sodium voltage-gated current (INa.TTX), TTX-sensitive sodium voltage-gated current (INa.TTXR), volume-sensitive chloride current (ICl.vol), voltage gated proton current (IHv), non-selective cation currents, besides mechanosensitive MG currents reported by Kamnkin et al. Manuscript describes single mechanically gated channels (MGCs), recorded simultaneously with whole cell MG currents. It demonstrates that cellular compression activates current, flowing through single MGCs (recorded in cell attached mode), along with whole cell MGC current (recorded in whole cell mode), which leads to generation of mechanically induced potentials (MIP). Cellular stretching inactivates those currents. Gd3+, cytochalasin D and colchicine inhibit both the whole-cell MG currents and single MG currents activity. All of those currents, which are mentioned above, together with MG currents contribute to alterations of fibroblast membrane potential (resting potentials and MIPs). Since fibroblasts are connected with cardiomyocytes via gap junctions, hyperpolarization of resting potential, triggered by cellular stretching, depolarization of the resting potentials, triggered by cellular compression, and the repolarization of the MIPs may potentially prolong the action potential duration in cardiomyocytes thereby predisposing the heart to arrhythmia.

An Anti-inflammatory Cytokine Interleukin-13: Physiological Role in the Heart and Mechanoelectrical Feedback

The role of cytokines in responses not associated with inflammation as well as their involvement in regulation of non-haematopoietic cell activity is intensively studied during last decades. Control of heart activity can be carried out by pro-inflammatory cytokines. During the last decades considerable attention was drawn to the role of cytokines in physiological reactions not related to inflammation as well as their involvement in regulation of non-haematopoietic cell activity. An involvement of pro-inflammatory cytokines in the control of heart activity is thoroughly discussed in many publications. On the other hand such experimental data for the anti-inflammatory cytokines are currently absent. This review briefly summarizes existing evidences of involvement of anti-inflammatory cytokine interleukin-13 in the control of heart functioning and presents our latest findings on IL-13 influence on cardiomyocytes activity. According to our data, application of the IL-13 led to moderate acute changes in electrical activity of cardiomyocytes. At the same time it did not cause any electrical abnormalities, which is opposite to inflammatory cytokines application effects. Application of IL-13 reduced the effect of the mechanical stretch application on electrical activity of cardiomyocytes. Negative inotropic effect of anti-inflammatory IL-13 contrasts with positive inotropic effect of most pro-inflammatory cytokines. Special attention is given to possible mechanisms of IL-13-signaling and its influence on cardiac function in norm and pathology.

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.