Topi Korhonen

Model of Excitation-Contraction Coupling of Rat Neonatal Ventricular Myocytes

The neonatal rat ventricular myocyte culture is one of the most popular experimental cardiac cell models. To our knowledge, the excitation-contraction coupling (ECC) of these cells, i.e., the process linking the electrical activity to the cytosolic Ca2+ transient and contraction, has not been previously analyzed, nor has it been presented as a complete system in detail. Neonatal cardiomyocytes are in the postnatal developmental stage, and therefore, the features of their ECC differ vastly from those of adult ventricular myocytes. We present the first complete analysis of ECC in these cells by characterizing experimentally the action potential and calcium signaling and developing the first mathematical model of ECC in neonatal cardiomyocytes that we know of. We show that in comparison to adult cardiomyocytes, neonatal cardiomyocytes have long action potentials, heterogeneous cytosolic Ca2+ signals, weaker sarcoplasmic reticulum Ca2+ handling, and stronger sarcolemmal Ca2+ handling, with a significant contribution by the Na+/Ca2+ exchanger. The developed model reproduces faithfully the ECC of rat neonatal cardiomyocytes with a novel description of spatial cytosolic [Ca2+] signals. Simulations also demonstrate how an increase in the cell size (hypertrophy) affects the ECC in neonatal cardiomyocytes. This model of ECC in developing cardiomyocytes provides a platform for developing future models of cardiomyocytes at different developmental stages.

Excitation-Contraction Coupling of the Mouse Embryonic Cardiomyocyte

In the mammalian embryo, the primitive tubular heart starts beating during the first trimester of gestation. These early heartbeats originate from calcium-induced contractions of the developing heart muscle cells. To explain the initiation of this activity, two ideas have been presented. One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca2+ release from intracellular stores initiating spontaneous intracellular calcium oscillations. We show with experiments that both of these mechanisms coexist and operate in mouse cardiomyocytes during embryonic days 9–11. Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats. This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat.

Mathematical model of mouse embryonic cardiomyocyte excitation-contraction coupling.

Excitation–contraction (E–C) coupling is the mechanism that connects the electrical excitation with cardiomyocyte contraction. Embryonic cardiomyocytes are not only capable of generating action potential (AP)-induced Ca2+ signals and contractions (E–C coupling), but they also can induce spontaneous pacemaking activity. The spontaneous activity originates from spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR), which trigger APs via the Na+/Ca2+ exchanger (NCX). In the AP-driven mode, an external stimulus triggers an AP and activates voltage-activated Ca2+ intrusion to the cell. These complex and unique features of the embryonic cardiomyocyte pacemaking and E–C coupling have never been assessed with mathematical modeling. Here, we suggest a novel mathematical model explaining how both of these mechanisms can coexist in the same embryonic cardiomyocytes. In addition to experimentally characterized ion currents, the model includes novel heterogeneous cytosolic Ca2+ dynamics and oscillatory SR Ca2+ handling. The model reproduces faithfully the experimentally observed fundamental features of both E–C coupling and pacemaking. We further validate our model by simulating the effect of genetic modifications on the hyperpolarization-activated current, NCX, and the SR Ca2+ buffer protein calreticulin. In these simulations, the model produces a similar functional alteration to that observed previously in the genetically engineered mice, and thus provides mechanistic explanations for the cardiac phenotypes of these animals. In general, this study presents the first model explaining the underlying cellular mechanism for the origin and the regulation of the heartbeat in early embryonic cardiomyocytes.

Modelling sarcoplasmic reticulum calcium ATPase and its regulation in cardiac myocytes.

When developing large-scale mathematical models of physiology, some reduction in complexity is necessarily required to maintain computational efficiency. A prime example of such an intricate cell is the cardiac myocyte. For the predictive power of the cardiomyocyte models, it is vital to accurately describe the calcium transport mechanisms, since they essentially link the electrical activation to contractility. The removal of calcium from the cytoplasm takes place mainly by the Na+/Ca2+ exchanger, and the sarcoplasmic reticulum Ca2+ ATPase (SERCA). In the present study, we review the properties of SERCA, its frequency-dependent and β-adrenergic regulation, and the approaches of mathematical modelling that have been used to investigate its function. Furthermore, we present novel theoretical considerations that might prove useful for the elucidation of the role of SERCA in cardiac function, achieving a reduction in model complexity, but at the same time retaining the central aspects of its function. Our results indicate that to faithfully predict the physiological properties of SERCA, we should take into account the calcium-buffering effect and reversible function of the pump. This ‘uncomplicated’ modelling approach could be useful to other similar transport mechanisms as well.

Regulation of excitation-contraction coupling in mouse cardiac myocytes: integrative analysis with mathematical modelling

BackgroundThe cardiomyocyte is a prime example of inherently complex biological system with inter- and cross-connected feedback loops in signalling, forming the basic properties of intracellular homeostasis. Functional properties of cells and tissues have been studied e.g. with powerful tools of genetic engineering, combined with extensive experimentation. While this approach provides accurate information about the physiology at the endpoint, complementary methods, such as mathematical modelling, can provide more detailed information about the processes that have lead to the endpoint phenotype.ResultsIn order to gain novel mechanistic information of the excitation-contraction coupling in normal myocytes and to analyze sophisticated genetically engineered heart models, we have built a mathematical model of a mouse ventricular myocyte. In addition to the fundamental components of membrane excitation, calcium signalling and contraction, our integrated model includes the calcium-calmodulin-dependent enzyme cascade and the regulation it imposes on the proteins involved in excitation-contraction coupling. With the model, we investigate the effects of three genetic modifications that interfere with calcium signalling: 1) ablation of phospholamban, 2) disruption of the regulation of L-type calcium channels by calcium-calmodulin-dependent kinase II (CaMK) and 3) overexpression of CaMK. We show that the key features of the experimental phenotypes involve physiological compensatory and autoregulatory mechanisms that bring the system to a state closer to the original wild-type phenotype in all transgenic models. A drastic phenotype was found when the genetic modification disrupts the regulatory signalling system itself, i.e. the CaMK overexpression model.ConclusionThe novel features of the presented cardiomyocyte model enable accurate description of excitation-contraction coupling. The model is thus an applicable tool for further studies of both normal and defective cellular physiology. We propose that integrative modelling as in the present work is a valuable complement to experiments in understanding the causality within complex biological systems such as cardiac myocytes.

Myogenic Skeletal Muscle Satellite Cells Communicate by Tunnelling Nanotubes

Quiescent satellite cells sit on the surface of the muscle fibres under the basal lamina and are activated by a variety of stimuli to disengage, divide and differentiate into myoblasts that can regenerate or repair muscle fibres. Satellite cells adopt their parents fibre type and must have some means of communication with the parent fibre. The mechanisms behind this communication are not known. We show here that satellite cells form dynamic connections with muscle fibres and other satellite cells by F‐actin based tunnelling nanotubes (TNTs). Our results show that TNTs readily develop between satellite cells and muscle fibres. Once developed, TNTs permit transport of intracellular material, and even cellular organelles such as mitochondria between the muscle fibre and satellite cells. The onset of satellite cell differentiation markers Pax‐7 and MyoD expression was slower in satellite cells cultured in the absence than in the presence of muscle cells. Furthermore physical contact between myofibre and satellite cell progeny is required to maintain subtype identity. Our data establish that TNTs constitute an integral part of myogenic cell communication and that physical cellular interaction control myogenic cell fate determination. J. Cell. Physiol. 223: 376–383, 2010.

Activation of Ca2+‐dependent protein kinase II during repeated contractions in single muscle fibres from mouse is dependent on the frequency of sarcoplasmic reticulum Ca2+ release

Aim:  To investigate the importance and contribution of calmodulin‐dependent protein kinase II (CaMKII) activity on sarcoplasmic reticulum (SR) Ca2+‐release in response to different work intensities in single, intact muscle fibres.

Automatic time-step adaptation of the forward Euler method in simulation of models of ion channels and excitable cells and tissue

Abstract The forward Euler method is commonly used to simulate models of ion channels and excitable cells and tissue. We show that the computational efficiency of these simulations could be significantly improved with automatic time-step adaptation. For this purpose a new easy-to-implement time-step adaptation method was developed.

Local Ca2+ Releases Enable Rapid Heart Rates in Developing Cardiomyocytes

Homogeneous intracellular Ca2+ release repeated with high frequency is the basis of the rhythmic contractions of cardiac myocytes. In adult ventricular myocytes, the t-tubular system enables transient homogeneous Ca2+ signals. Interestingly, the developing cardiomyocytes do not have t-tubuli and Ca2+ signal propagation in the cytosol is based on the relatively slow diffusion of Ca2+ ions. This is likely to result in spatiotemporal heterogeneity of Ca2+, which limits the maximal frequency of the Ca2+ signals. We observed that intracellular Ca2+ signals of 12.5 days old mouse embryonic ventricular myocytes are more homogeneous than expected if the Ca2+ signals would propagate by pure diffusion. To study the propagation more accurately, we injected a small amount of Ca2+ to a single point in the cytosol via patch-clamp pipette while performing the line-scan imaging of the intracellular Ca2+. With this method we found that inhibition of the sarcoplasmic reticulum (SR) Ca2+ release channels results in 3-fold slowing of Ca2+ signal propagation (control: 10.1 ± 2.7 ms/μm vs. ryanodine (50 μM): 33.6 ± 9.2 ms/μm, P < 0.05). This suggested that the propagation of Ca2+ signals is amplified with local SR Ca2+ releases. Immunolabeling of SR Ca2+ release and uptake proteins revealed a regular structure throughout the cytosol at ∼2 μm intervals. These extensions of SR were equally functional in all parts of the cytosol. To further study the role of these local Ca2+ release sites in developing cardiomyocytes, we implemented a model of them into the previously published mathematical model of an embryonic cardiomyocyte. The computer simulations showed that the local Ca2+ releases are prerequisite for synchronizing the global intracellular Ca2+ releases upon electrical excitation and maintaining the capability of developing cardiomyocytes to generate spontaneous pacemaking at a sufficiently high frequency.

It Takes Two to Tango: Regulation of Sarcoplasmic Reticulum Calcium ATPase by CaMK and PKA in a Mouse Cardiac Myocyte

At the cellular level, one of the most important regulators of myocardial relaxation is the sarcoplasmic reticulum calcium ATPase (SERCA), which is controlled by phosphorylation both directly and indirectly through the endogenous inhibitor phospholamban. According to experimental observations, this regulation is interplay of protein kinase A (PKA), and calcium/calmodulin dependent kinase II (CaMK). Despite the physiological importance of this modulation, the significance of the crosstalk in this dual regulation has not been thoroughly described, and the exact quantitative roles of these two enzymes have remained partly elusive. We use the approach of mathematical modeling to dissect the different aspects of the regulation of SERCA in cardiomyocytes. We present a novel model of SERCA that includes phosphorylation targets for both PKA and CaMK. To study the physiological impact of these regulatory mechanisms, we implement the SERCA model into a mathematical model of a mouse ventricular myocyte. We validate the model by comparing the simulated results to the corresponding in vivo observations, both in physiological phenomena and transgenic test cases. Our results show that under varying levels of beta-adrenergic stimulation, and thus at varying PKA activities, the frequency-dependent changes in the calcium dynamics show a clear dose-dependence. Furthermore, the in silico experiments indicate that these changes are drastically blocked with CaMK inhibitors. The results also point out the diverging time windows of regulation for PKA (∼ tens of seconds) and CaMK (∼ few minutes). Based on the results, we conclude that despite the prominent role of PKA in the beta-adrenergic modulation of calcium dynamics in a cardiac myocyte, both the direct and the indirect effects mediated by CaMK phosphorylation appear to more important for the regulation of SERCA, though the time scale of observation has a significant impact on the quantitative roles of these two enzymes.