Intracellular Calcium Dynamics Model in Rabbit Ventricular Myocytes with Realistic Transverse Tubules
Rabbit ventricular myocytes are often used to study heart diseases. Dr. Frank Sachse’s group at the University of Utah has been developing a technique to provide submicrometer resolution images of living ventricular myocytes. The transverse tubular system in these cells consists of cell membrane invaginations (t-tubules) that are essential for efficient cardiac excitation-contraction coupling, in other words, the beating of the heart. The t-system often exhibits morphological changes associated with cardiac development, hypertrophy and heart failure. To further understand how t-tubule micro-anatomy, L-type Ca2+ channel (LCC) clustering, and allosteric activation of Na+/ Ca2+ exchanger (NCX) by L-type Ca2+ current affects intracellular Ca2+ dynamics, a group of researchers led by Dr. Pete Kekenes-Huskey and Anushka Michailova from NBCR has collaborated with Dr. Sachse to build a computational model of a single t-tubule with a realistic geometry obtained through confocal microscopy. The model also includes the surrounding half-sarcomeres for rabbit ventricular myocytes. The model was refined using the NBCR tool GAMer plugin to the popular Blender software package. Blender has been used in films such as “Spiderman 2”, and was used by NBCR researchers to develop a 3D research tool called BLAMer (Blender + GAMer). Through the BLAMer interface, a mesh structure that represents the t-tubule is marked, refined, and smoothened so that it is suitable for finite element method based simulations.
Using a series of coupled reaction-diffusion equations to describe the spatio-temporal concentration profiles of free and buffered intracellular Ca2+, and parameters from voltage-clamp protocols of L-type Ca2+ current and line-scan recordings of Ca2+ concentration profiles in rabbit cells, Kekenes-Huskey and colleagues performed simulations using an improved version of the NBCR software package SMOL (See Fig. 1). The new SMOL package is able to solve multiple coupled partial differential equations with non-linear ordinary equations.
The simulation results are in excellent agreement with experimental measurements of global Ca2+ transient in myocytes loaded with 50 μM Fluo-3, a fluorescent calcium buffer and indicator. The local Ca2+ concentrations within the cytosol and sub-sarcolemma, as well as the local trigger fluxes of Ca2+ crossing the cell membrane, are sensitive to details of t-tubule micro-structure and membrane Ca2+ flux distribution. Moreover, the model predicts that local Ca2+ trigger fluxes are at least threefold to eightfold higher than the whole-cell Ca2+ trigger flux. We found also that the activation of allosteric Ca2+-binding sites on the NCX could provide a mechanism for regulating global and local Ca2+ trigger fluxes in vivo.
In summary, this study shows that improved structural and functional models could improve our understanding of the contributions of L-type and NCX fluxes to intracellular Ca2+ dynamics. In the long term, such studies are important in the development of new therapeutic techniques for heart diseases.
NBCR works with collaborators such as Profs. Sachse and Bridge to carry out multiscale modeling. Algorithms and tools developed as part of this project are available for download at nbcr.ucsd.edu.
- Kekenes-Huskey, P.M.; Cheng, Y.; Hake, J.E.; Sachse, F.B.; Bridge, J.H.; Holst, M.J.; McCammon, J.A.; McCulloch, A.D.; Michailova, A.P., Modeling effects of L-type Ca2+ current and Na+-Ca2+ exchanger on Ca2+ trigger flux in rabbit myocytes with realistic t-tubule geometries. Frontiers in Computational Physiology and Medicine: Calcium Signaling in Cardiac Myocyte 2012, 3, 351, PMC3463892 T0002.
Researchers: NBCR: Pete Kekenes-Huskey, Michael Holst, Andy McCammon, Andrew McCulloch, Anushka Michailova; Collaborative Researchers: Johan Hake (Simula), Frank Sachse (University of Utah), John Bridge (University of Utah)
Figure 1: Model geometry and diagram illustrating Ca2+ dynamics in rabbit ventricular myocytes with a pharmacologically disabled sarcoplasmic reticulum.
- (a) Cardiac sarcolemma visualized using scanning, confocal microscopy and labeled using 3D graphics. Localized aggregates of L-type Ca2+ channels (red spots).
- (b) T-tubule mesh and its surrounding half-sarcomere.
- (c) Schematic drawing of Ca2+ entry and extrusion via the sarcolemma and Ca2+ buffering and diffusion inside the myocyte. The line-scan is positioned at 200 nm away from the t-tubule mouth (yellow line and yellow spot in (b)).