Modeling of mechanoelectric coupling in cardiomyocytes in norm and pathology

We developed mathematical models of the electromechanical function of cardiomyocytes and the simplest mechanically heterogeneous myocardial systems, muscle duplexes. By means of these models we studied the contribution of mechanoelectric feedbacks to the contractile activity of the myocardium in norm and pathology. In particular, we simulated and clarified the effects of mechanical conditions on both the form and the duration of the action potential during contractions. From this standpoint different kinds of myocardium mechanical heterogeneity were analyzed. As we have established, the latter can play both a positive and a negative role, depending on the distribution of mechanical nonuniformity and the sequence of activation of heterogeneous myocardium system elements. By means of the same models, we studied the contribution of mechanical factors to the arrhythmogenicity in the case of the cardiomyocyte calcium overload caused by the attenuation of the sodium-potassium pump and outlined the ways for correcting the contractile function in these disturbances.     

Mathematical models for the study of electromechanical and mechanoelectrical coupling in the myocardium

Mathematical models have been developed to describe interactions of electrical, mechanical and chemical processes in cardiomyocytes. The models simulate wide range of experimental data on excitation-contraction coupling and, more importantly, on mechanoelectric feedback in heart muscle. The model results clearly show that mechano-dependence of intracellular calcium handling due to cooperative effects of contractile proteins activation plays a key role in cardiac mechanoelectric coupling. At the same time, mechanosensitive currents can also contribute to action potential responses to mechanical perturbations. Using this model to study the heterogeneous myocardium we have shown that temporal and functional electromechanical heterogeneity of coupled cardiomyocytes can essentially determine the myocardium contractility. Optimization of the electromechanical function of contractile system emerges from the fine coordination between the activation sequence of cardiomyocytes, their local electromechanical properties and the mechanical interaction during contraction.     

Contribution of mechanical factors to arrhythmogenesis in calcium overloaded cardiomyocytes: model predictions and experiments

It is well-known that Ca²⁺ overload in cardiomyocytes may underlie arrhythmias. However, the possible contribution of mechanical factors to rhythm disturbances in Ca²⁺ overloaded myocytes has not been sufficiently investigated. We used a mathematical model of the electrical and mechanical activity of cardiomyocytes to reveal an essential role of the mechanisms of cardiac mechano-electric feedback in arrhythmogenesis in Ca²⁺ overloaded myocardium. In the model, the following mechanical factors increased Ca²⁺ overload in contracting cardiomyocytes and promoted rhythm disturbances: i) a decrease in the mechanical load for afterloaded contractions; and ii) a decrease in the initial length of sarcomeres for isometric twitches. In exact accordance with the model predictions, in experiments on papillary muscles from the right ventricle of guinea pigs with Ca²⁺ overloaded cardiomyocytes (using 0.5-1 μM of ouabain), we found that emergence of rhythm disturbances and extrasystoles depends on the mechanical conditions of muscle contraction.     

Electromechanical heterogeneity of the myocardium

Herein we discuss modem data showing that ventricle's working myocardium is highly heterogeneous. Significant transmural differences in electrophysiological and biomechanical properties of cardiomyocytes are reviewed. The reviewed evidence of myocardial heterogeneity constitutes the basis for modem assessment of segmental kinetics of different regions in intact heart. We used muscle duplexes as condensed models of a heterogeneous myocardial system. Experimental data, presented here were obtained both in biological duplexes formed by isolated myocardial preparations and in mathematical models of muscle duplexes. We showed that specific functional heterogeneity of cardiomyocytes, related to their excitation sequence, allowed the myocardium to optimise its contractile function and smooth dispersion of repolarisation.     

Ultracytochemical heterogeneity of cytochrome oxidase activity in rat cardiomyocytes

Optimum conditions are developed for ultracytochemical detection of cytochrome oxidase in cardiomyocytes from the rat heart ventricles. Conditions mentioned above rest on using of o-dianisidine and double contrasting of microscopic sections by uranyl and lead acetates. Data from studies in heterogeneity of cytochrome oxidase activity as dependent on the structural-functional state (electronic density) of myocardium cells are presented.     

Cardiomyocyte survival and DNA repair in myocardium from C57Bl/6 and mdx mice after dynamical stress

Mdx mice cardiomyocytes are a perspective model to study survival of terminally differentiated cardiomyocytes and formation of cardiomyopathy under conditions of oxidative stress. It was previously observed that dynamical stress induced formation of low molecular DNA fragments. It is beyond question that DNA fragmentation develops because of formation of double strand DNA breaks (DNA DSB). To record appearance and disappearance of DNA DSB we used antibodies to phosphorylated histone H2Ax (histone gamma-H2Ax.). The presence of DNA DSB was estimated in 0.05% and 6.7% of cardiomyocytes in the myocardium form C57B1 and mdx mice without stress, respectively. The part of cardiomyocytes with DNA DSB increased in an hour after stress up to 1.0% and 41.7% in C57B1 and mdx mice, respectively. In 24 h after stress, the myocardium from mdx mice contained 5.2% of gamma-H2Ax-positive cardiomyocytes and no C57B1 myocardium was found with any amount of gamma-H2Ax-positive cells. The results presented show induction of DNA damage by dynamical stress and restoration of normal DNA structure in the cells of both strains in 24 h after stress. There was no mdx mice death after used dynamical stress. To estimate the real contribution of DNA repair to the survival of cardiomyocytes we have counted the cardiomyocyte loss. Morphometric analysis demonstrated that cell concentration in myocardium from mdx mice under normal conditions was less than that one in myocardium of C57B1/6. The cell loss varied between 20% for the base and 40% for the apex of mdx mice hearts. In 24 h after stress, the cell loss in the myocardium of mdx mice amounted to 2.5%. The difference between the number of cells with damaged DNA structure and the index of the real cell loss allows concluding that DNA repair makes a real contribution to the survival of mdx mice cardiomyocytes after dynamical stress.     

Pan-epicardial lineage tracing reveals that epicardium derived cells give rise to myofibroblasts and perivascular cells during zebrafish heart regeneration

Myocardial infarction (MI) leads to a severe loss of cardiomyocytes, which in mammals are replaced by scar tissue. Epicardial derived cells (EPDCs) have been reported to differentiate into cardiomyocytes during development, and proposed to have cardiomyogenic potential in the adult heart. However, mouse MI models reveal little if any contribution of EPDCs to myocardium. In contrast to adult mammals, teleosts possess a high myocardial regenerative capacity. To test if this advantage relates to the properties of their epicardium, we studied the fate of EPDCs in cryoinjured zebrafish hearts. To avoid the limitations of genetic labelling, which might trace only a subpopulation of EPDCs, we used cell transplantation to track all EPDCs during regeneration. EPDCs migrated to the injured myocardium, where they differentiated into myofibroblasts and perivascular fibroblasts. However, we did not detect any differentiation of EPDCs nor any other non-cardiomyocyte population into cardiomyocytes, even in a context of impaired cardiomyocyte proliferation. Our results support a model in which the epicardium promotes myocardial regeneration by forming a cellular scaffold, and suggests that it might induce cardiomyocyte proliferation and contribute to neoangiogenesis in a paracrine manner.     

Electromechanical coupling in cardiomyocytes from transmural layers of guinea pig left ventricle

The electrical and mechanical activity of heart ventricle cardiomyocytes is known to vary depending on the spatial location of cells in the wall, in particular, transmurally from the sub-endocardial layer to the sub-epicardial one. To investigate intracellular mechanisms of the functional heterogeneity of cardiomyocytes we developed mathematical models of the electromechanical coupling in cardiomyocytes from different transmural layers across the left ventricle (LV) wall of guinea pig. It is shown that the mechanisms of both direct linkages and feedback in the electromechanical coupling contribute to differences in both the shape and duration of action potential, and speed characteristics of contraction between isolated cardiac myocytes from the sub-endocardial and sub-epicardial layers.     

Immunolocalization of interleukin-1 receptor antagonist in healthy and infarcted myocardium

Ischemic heart disease is a widespread cause of death. During infarction, myocardial injury is mediated by release of several pro-inflammatory cytokines including multifunctional interleukin-1 (IL-1). In various tissues, IL-1-mediated deleterious effects are known to be attenuated via the over-expression of natural anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra). In the present investigation, IL-1ra distribution in healthy and infarcted myocardium was studied by light and electron microscopy. After immunostaining, weak positivity resulted for cardiomyocytes in normal myocardium and, at higher degrees, in infarction border areas and ischemic ones. In ischemic areas, additional reactivity was displayed by the extracellular matrix and intravascular plasma. Immunogold labelling provided further details on intracytoplasmatic and extracellular distribution; in particular, noticeable gold particle distribution appeared on intercalated discs in normal and hypertrophic cardiomyocytes, as well as on thickened Z-lines for these latter. The present results suggest that cardiomyocytes represent a major source of IL-1ra in vivo, even though additional contribution by blood derived IL-1ra is to be taken in account in ischemic areas. In addition, ischemia-associated intracytoplasmic IL-1ra increase and its additional presence in the extracellular matrix is consistent with the concept that this cytokine plays a cardioprotective role at different levels and by distinct mechanisms.     

Mathematical Modeling of Mechanically Modulated Rhythm Disturbances in Homogeneous and Heterogeneous Myocardium with Attenuated Activity of Na<Superscript>+</Superscript>–K<Superscript>+</Superscript> Pump

A mathematical model of the cardiomyocyte electromechanical function is used to study contribution of mechanical factors to rhythm disturbances in the case of the cardiomyocyte calcium overload. Particular attention is paid to the overload caused by diminished activity of the sodium-potassium pump. It is shown in the framework of the model, where mechano-calcium feedback is accounted for that myocardium mechanics may significantly enhance arrhythmogenicity of the calcium overload. Specifically, a role of cross-bridge attachment/detachment processes, a role of mechanical conditions of myocardium contractions (length, load), and a role of myocardium viscosity in the case of simulated calcium overload have been revealed. Underlying mechanisms are analyzed. Several approaches are designed in the model and compared to each other for recovery of the valid myocardium electrical and mechanical performance in the case of the partially suppressed sodium-potassium pump.     

Functional heterogeneity of the atrial myocardium in patients with congenital and acquired heart defects]

In experiments on atrium trabeculae the heterogeneity of myocardium contractile activity in patients operated on for inborn or acquired heart defects was studied. Contractile activity was assessed in isometric regime of muscle drugs work. The degree of functional heterogeneity was reported to differ in myocardium biopsies from patients with inborn or acquired heart defects. The difference was expressed in susceptibility to stimulating action or electrical impulses and in the degree of the change of myocardium contractile activity. The study of human myocardium functional heterogeneity is likely to present a new approach to increase efficacy of the work of pathologically changed heart muscle.     

Role of myocardial viscoelasticity in disturbances of electrical and mechanical activity in calcium overloaded cardiomyocytes: mathematical modeling

Cardiomyocyte Ca²⁺ overload is closely linked to cardiac arrhythmias. We have earlier shown in a mathematical model that myocardium mechanical activity may contribute to rhythm disturbances induced by Ca²⁺ overload in cardiomyocytes with reduced Na⁺-K⁺ pump work (Sulman et al., 2008). The same model is used here to address possible contribution of the passive mechanical properties of cardiac muscle (i.e. myocardial viscous and elastic properties) to the arrhythmogenesis. In a series of contractions at regular pacing rate of 75 beats/min a model with higher viscosity demonstrated essentially earlier appearance of extrasystoles due to a faster cardiomyocyte Ca²⁺ loading up to a level triggering spontaneous Ca²⁺ releases from the sarcoplasmic reticulum. The model predicts that myocardial elasticity also may affect arrhythmogenesis in cardiomyocytes overloaded with Ca²⁺. Contribution of the mechanical properties of the myocardial tissue to the arrhythmia has been analyzed for wide ranges of both viscosity and elasticity coefficients. The results suggest that myocardial viscoelastic properties may be a factor affecting Ca²⁺ handling in cardiomyocytes and contributing to cardiac mechano-electric feedback in arrhythmogenesis.     

Ultrastructural mechanisms of myocardial atrophy in white rats during starvation

By means of complex morphological, morphometrical and stereological analyses the myocardium of Wistar rats was studied in full alimentary starvation during 6 days. The availability of two mechanisms of myocardial atrophy in adaptation to full starvation was revealed, these are the diminution of dimensions of parenchymatous elements and the decrease of muscle cell number. By ultrastructural investigation of cardiomyocytes the signs of structural protein synthesis decline were revealed, this is a simple myocardial atrophy. In quantitative analysis of cardiomyocytes the decrease of their number without changes in proportions of cells with different nuclei number was observed, that indicated systematic character of muscle cell elimination out of myocardium by apoptosis mechanism involvement, this is a numerical myocardial atrophy. Stereological analysis of myocardial atrophy development in conditions of full starvation determined the main regulated indices, these are the absolute total myofibril mass and mitochondrial mass and surface area.     

Upregulation of corin gene expression in hypertrophic cardiomyocytes and failing myocardium

High levels of plasma atrial natriuretic peptides (ANP) are associated with pathological conditions such as congestive heart failure (CHF). Recently, we have identified a cardiac serine protease, corin, that is the pro-ANP convertase. In this study, we examined the regulation of corin gene expression in cultured hypertrophic cardiomyocytes and in the left ventricular (LV) myocardium of a rat model of heart failure. Quantitative RT-PCR analysis showed that both corin and ANP mRNA levels were significantly increased in phenylephrine (PE)-stimulated rat neonatal cardiomyocytes in culture. The increase in corin mRNA correlated closely with the increase in cell size and ANP mRNA expression in the PE-treated cells (r = 0.95, P < 0.01; r = 0.92, P < 0.01, respectively). The PE-treated cardiomyocytes had an increased activity in converting recombinant human pro-ANP to biologically active ANP, as determined by a pro-ANP processing assay and a cell-based cGMP assay. In a rat model of heart failure induced by ligation of the left coronary artery, corin mRNA expression in the noninfarcted LV myocardium was significantly higher than that of control heart tissues from sham-operated animals, when examined by Northern blot analysis and RT-PCR at 8 wk. These results indicate that the corin gene is upregulated in hypertrophic cardiomyocytes and failing myocardium. Increased corin expression may contribute to elevation of ANP in the setting of cardiac hypertrophy and heart failure.     

Importance of endogenous substrates for cultured adult rat cardiac myocytes

In Ca-tolerant adult cardiomyocytes the contribution of endogenous substrates (glycogen, tri- and diacylglycerol) to oxidative substrate metabolism was investigated. After 4 h in culture medium (M 199 plus 4% fetal calf serum) the cellular triacylglycerol content is 3.6-fold higher than in fresh myocardium and reflects the free fatty acid composition of the medium. When triacylglycerol is degraded, all long-chain fatty acids are hydrolysed at equal rates. In these quiescent cells, the activity of pyruvate dehydrogenase is low (10% of full activity, in Tyrode solution with 5 mM glucose). Up to 30% of full pyruvate dehydrogenase activity, the contribution of non-lipid substrates (glycogen, glucose, lactate and pyruvate) to oxidative energy production is correlated to pyruvate dehydrogenase activity. At 5 mM medium concentration, glucose, lactate and pyruvate share in energy production the proportions of 15, 36 and 50%, whereas endogenous lipolysis accounts for 78, 61 and 46%. It is concluded that these quiescent cardiomyocytes represent cardiac metabolism in a basal state in which the preference for fatty acids, especially from endogenous lipids, is very pronounced. The utilization of endogenous substrates therefore has to be considered in all studies investigating the oxidative metabolism of these isolated cells.     

Mechano-electric interactions in heterogeneous myocardium: development of fundamental experimental and theoretical models

The heart is structurally and functionally a highly non-homogenous organ, yet its main function as a pump can only be achieved by the co-ordinated contraction of millions of ventricular cells. This apparent contradiction gives rise to the hypothesis that ‘well-organised’ inhomogeneity may be a pre-requisite for normal cardiac function. Here, we present a set of novel experimental and theoretical tools for the study of this concept. Heterogeneity, in its most condensed form, can be simulated using two individually controlled, mechanically interacting elements (duplex). We have developed and characterised three different types of duplexes: (i) biological duplex, consisting of two individually perfused biological samples (like thin papillary muscles or a trabeculae), (ii) virtual duplex, made-up of two interacting mathematical models of cardiac muscle, and (iii) hybrid duplex, containing a biological sample that interacts in real-time with a virtual muscle. In all three duplex types, in-series or in-parallel mechanical interaction of elements can be studied during externally isotonic, externally isometric, and auxotonic modes of contraction and relaxation. Duplex models, therefore, mimic (patho-)physiological mechano-electric interactions in heterogeneous myocardium at the multicellular level, and in an environment that allows one to control mechanical, electrical and pharmacological parameters. Results obtained using the duplex method show that: (i) contractile elements in heterogeneous myocardium are not ‘independent’ generators of tension/shortening, as their ino- and lusitropic characteristics change dynamically during mechanical interaction—potentially matching microscopic contractility to macroscopic demand, (ii) mechanical heterogeneity contributes differently to action potential duration (APD) changes, depending on whether mechanical coupling of elements is in-parallel or in-series, which may play a role in mechanical tuning of distant tissue regions, (iii) electro-mechanical activity of mechanically interacting contractile elements is affected by their activation sequence, which may optimise myocardial performance by smoothing intrinsic differences in APD. In conclusion, we present a novel set of tools for the experimental and theoretical investigation of cardiac mechano-electric interactions in healthy and/or diseased heterogeneous myocardium, which allows for the testing of previously inaccessible concepts.     

The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion

Natural models of heart regeneration in lower vertebrates such as zebrafish are based on invasive surgeries causing mechanical injuries that are limited in size. Here, we created a genetic cell ablation model in zebrafish that facilitates inducible destruction of a high percentage of cardiomyocytes. Cell-specific depletion of over 60% of the ventricular myocardium triggered signs of cardiac failure that were not observed after partial ventricular resection, including reduced animal exercise tolerance and sudden death in the setting of stressors. Massive myocardial loss activated robust cellular and molecular responses by endocardial, immune, epicardial and vascular cells. Destroyed cardiomyocytes fully regenerated within several days, restoring cardiac anatomy, physiology and performance. Regenerated muscle originated from spared cardiomyocytes that acquired ultrastructural and electrophysiological characteristics of de-differentiation and underwent vigorous proliferation. Our study indicates that genetic depletion of cardiomyocytes, even at levels so extreme as to elicit signs of cardiac failure, can be reversed by natural regenerative capacity in lower vertebrates such as zebrafish.     

Overexpression of the NHE1 isoform of the Na+/H+ exchanger causes elevated apoptosis in isolated cardiomyocytes after hypoxia/reoxygenation challenge

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH in the myocardium and other tissues. NHE1 is an important mediator of myocardial damage that occurs after ischemia–reperfusion injury. It has also been implicated in apoptotic damage in many tissues and its expression and activity are elevated in disease states in the myocardium. In this study, we examined the effect of additional exogenous NHE1 expression on isolated cardiomyocytes susceptibility to ischemia/reperfusion damage. Exogenous NHE1 elevated Na⁺/H⁺ exchanger expression and activity when introduced into isolated cardiomyocytes through an adenoviral system. Isolated cardiomyocytes were subjected to simulated ischemia and reperfusion after infection with either control or NHE1-containing adenovirus. Cells were placed into an anaerobic chamber and effects of NHE1 expression after hypoxia/reoxygenation were examined. Hypoxia/reoxygenation increased caspase-3-like activity in controls, and the effect was greatly magnified in cells expressing NHE1 protein. It also elevated the percentage of apoptotic cardiomyocytes, which was also aggravated by expression of NHE1 protein. Hypoxia/reoxygenation also increased phospho-ERK levels. Elevated NHE1 expression was coincidental with increased expression of the ER stress protein, protein disulfide isomerase (PDI) and calreticulin (CRT). Our results demonstrate that increased NHE1 protein expression makes cells more susceptible to damage induced by hypoxia/reoxygenation in isolated cardiomyocytes. They suggest that elevated NHE1 in cardiovascular disease could predispose the human myocardium to enhanced apoptotic damage.     

Ischemic and hypoxic preconditioning protect cardiac muscles via intracellular ROS signaling

Oxidative stress can cause extensive damage to cardiac tissue under reperfusion conditions. However, preconditioning the myocardium may diminish these negative effects and alleviate reperfusion injury. There are a variety of preconditioning therapies, such as ischemic preconditioning (IPC) and hypoxic preconditioning (HPC), each targeting specific channels, receptors, and/or intracellular molecules. Ischemic preconditioning involves brief periods of ischemia followed by brief periods of reperfusion, thus strengthening the cardiac resistance for a longer period of ischemia. IPC involves complex mechanisms, some of which are still not completely understood today. Nevertheless, many studies have already established models of IPC. In addition, similar to IPC, HPC has also been recognized as preventing reperfusion injury. Reactive oxygen species (ROS) are known mediators of IPC and HPC. Particularly, mitochondria-generated ROS initiate activity of several beneficial preconditioning pathways. The role of ROS is paradoxical; low levels of ROS are key factors in signaling IPC/HPC, but high levels of ROS can contribute to increased oxidative stress on cardiomyocytes. Therefore, it is important to determine the molecular mechanism of IPC and HPC to avoid excessive accumulation of ROS to prevent cardiac injury. In this review, we will outline IPC and HPC, explaining the putative role of ROS in both pathways. We will also discuss preconditioning efficacy in certain conditions such as exercise and how the aging myocardium responds to preconditioning therapies.     

Stretch-induced hypertrophy activates NFkB-mediated VEGF secretion in adult cardiomyocytes

Hypertension and myocardial infarction are associated with the onset of hypertrophy. Hypertrophy is a compensatory response mechanism to increases in mechanical load due to pressure or volume overload. It is characterized by extracellular matrix remodeling and hypertrophic growth of adult cardiomyocytes. Production of Vascular Endothelial Growth Factor (VEGF), which acts as an angiogenic factor and a modulator of cardiomyocyte function, is regulated by mechanical stretch. Mechanical stretch promotes VEGF secretion in neonatal cardiomyocytes. Whether this effect is retained in adult cells and the molecular mechanism mediating stretch-induced VEGF secretion has not been elucidated. Our objective was to investigate whether cyclic mechanical stretch induces VEGF secretion in adult cardiomyocytes and to identify the molecular mechanism mediating VEGF secretion in these cells. Isolated primary adult rat cardiomyocytes (ARCMs) were subjected to cyclic mechanical stretch at an extension level of 10% at 30 cycles/min that induces hypertrophic responses. Cyclic mechanical stretch induced a 3-fold increase in VEGF secretion in ARCMs compared to non-stretch controls. This increase in stretch-induced VEGF secretion correlated with NFkB activation. Cyclic mechanical stretch-mediated VEGF secretion was blocked by an NFkB peptide inhibitor and expression of a dominant negative mutant IkBα, but not by inhibitors of the MAPK/ERK1/2 or PI3K pathways. Chromatin immunoprecipitation assays demonstrated an interaction of NFkB with the VEGF promoter in stretched primary cardiomyocytes. Moreover, VEGF secretion is increased in the stretched myocardium during pressure overload-induced hypertrophy. These findings are the first to demonstrate that NFkB activation plays a role in mediating VEGF secretion upon cyclic mechanical stretch in adult cardiomyocytes. Signaling by NFkB initiated in response to cyclic mechanical stretch may therefore coordinate the hypertrophic response in adult cardiomyocytes. Elucidation of this novel mechanism may provide a target for developing future pharmacotherapy to treat hypertension and heart disease.