bioRxiv preprint doi: https://doi.org/10.1101/2020.08.13.249144; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 2+ 2+ Ca release via IP3Rs increases RyR mediated Ca spark frequency in ventricular cardiomyocytes without altering spark amplitude and duration Agn_eTil¯unait_e1,4, David Ladd1,4,5, Hilary Hunt1,4, Christian Soeller3, H. Llewelyn Roderick2, Edmund J. Crampin1,5,y, Vijay Rajagopal4,y,* 1 Systems Biology Laboratory, School of Mathematics and Statistics, University of Melbourne, Australia 2 Laboratory of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium 3 Living Systems Institute, University of Exeter, Exeter EX4 4QD, UK 4 Cell Structure and Mechanobiology Group, Department of Biomedical Engineering, Melbourne School of Engineering, University of Melbourne, Australia 5 ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, School of Chemical and Biomedical Engineering, University of Melbourne, Australia y These authors contributed equally to the supervision of this work. * Corresponding author Email: [email protected] Abstract Calcium plays critical roles in cardiac cells, coupling electrical excitation to mechanical contraction with each heartbeat, while simultaneously mediating biochemical signals that regulate cell growth. While ryanodine receptors (RyRs) are fundamental to generation of elementary calcium release events (sparks) and global calcium elevations that underlie excitation-contraction coupling (ECC), calcium release via inositol 1,4,5-trisphosphate receptors (IP3Rs) is also reported in cardiomyocytes. IP3R calcium release modifies ECC as well as contributing to downstream regulation of hypertrophic gene expression. Recent studies suggest that proximal localisation of IP3Rs with RyRs contributes to their ability to modify Ca2+ handling during ECC. Here we aim to 2+ determine the mechanism by which IP3Rs modify Ca handling in cardiomyocytes. We develop a mathematical model incorporating the stochastic behaviour of receptor opening that allows for the parametric tuning of the system to reveal the impact of IP3Rs on spark activation. By testing multiple spark initiation mechanisms, we find 2+ that Ca release via IP3Rs result in increased propensity for spark initiation within 2+ the cardiac dyad. Our simulations suggest that opening of IP3Rs elevates Ca within the dyad, which increase the probability of spark initiation. Finally, we find that while increasing the number of IP3Rs increases the probability of spark formation, it has little effect on spark amplitude, duration, or overall shape. Our study therefore suggests that 2+ IP3R play a critical role in modulating Ca signaling for excitation contraction coupling Author summary While Ca2+ release through ryanodine receptors (RyRs) initiates contraction in cardiomyocytes, the role of inositol 1,4,5-trisphosphate receptors (IP3Rs ) in August 13, 2020 1/17 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.13.249144; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. cardiomyocytes is less clear with Ca2+ release through these channels being invoked in regulating ECC and hypertrophic signalling. RyRs generate cytosolic Ca2+ signals 2+ through elemental Ca release events called sparks. The mechanisms by which IP3Rs influence cytosolic Ca2+ are not well understood. We created a 1D model of calcium spark formation in a cardiomyocyte dyad|the primary site of elemental RyR-based calcium release. We investigated possible behaviours of IP3Rs and their interaction with 2+ RyRs in generating Ca sparks. We show that for high IP3 concentration, a large number of IP3Rs and high IP3R affinity are required to noticeably affect spark shape. 2+ At lower IP3 concentration IP3Rs can increase Ca spark activity, but do not significantly alter the spark shape. Finally our simulations suggest that spark frequency 2+ can be reliably increased when IP3Rsactivity is such that a small continuous Ca flux is introduced to the dyad to elevate Ca2+ , and not via brief but high Ca2+ release from these receptors. Introduction 1 2+ Calcium plays a fundamentally important role in the regulation of each heartbeat. Ca 2 2+ flux through L-type Ca channels (LTCC) couples electrical activation to muscle 3 contraction through excitation-contraction coupling (ECC) [1,2]. Membrane 4 depolarisation triggers a small influx of calcium through voltage-gated LTCCs (Fig. 1A) 5 into 10-15 nm wide calcium microdomains [3,4] called dyads. In these regions, LTCC 6 2+ channels are juxtaposed with a set of Ca channels called ryanodine receptors (RyRs) 7 on the intracellular sarcoplasmic reticulum (SR) compartment. RyRs are sensitised by 8 2+ 2+ 2+ the Ca influx and release a larger amount of Ca from the SR. This local Ca 9 release event at the dyad is known as a calcium spark (Fig. 1B) and cardiac contraction 10 2+ is determined by the sum of these elementary local Ca release events at the many 11 dyads regularly distributed through the cardiomyocyte volume. 12 2+ In a majority of cell types, neurohormonal stimulation of intracellular Ca is 13 mediated by inositol 1,4,5-trisphosphate (IP3 ) and IP3 receptors (IP3Rs) located on the 14 endoplasmic reticulum (ER) [5]. In cardiomyocytes, activation of G-protein coupled 15 receptors (GPCR) for neurotransmitters such as endothelin-1 (ET-1) and angiotensin-II 16 2+ (AngII) leads to elevation of IP3 and Ca release via IP3Rs [6{9]. Crosstalk between 17 IP3Rs and RyRs, in which activation of IP3Rs via elevated IP3 leads to recruitment of 18 2+ neighbouring RyRs receptors, leading to larger Ca release, was first suggested in 19 smooth muscle cells [10]. Similar interaction between IP3Rs and RyRs has also been 20 proposed in embryonic myocytes [11, 12], atrial cardiac myocytes [13{15], spontaneously 21 hypertensive rat (SHR) myocytes [8], and rabbit ventricular myocytes [16]. Given the 22 2+ lower abundance and Ca flux of IP3Rs relative to RyRs, how they can affect ECC 23 remains poorly understood. In particular, the characteristics of IP3R gating that may 24 lead to recruitment of RyR receptors also remains to be determined. 25 2+ Experimental investigations [17] and computational models [18,19] of Ca sparks 26 have elucidated the role of the spatial distribution and density of RyR channels, and 27 2+ their stochastic interactions, in determining the spatio-temporal characteristics of Ca 28 sparks in health and disease [20]. IP3Rs are proposed to collocate with 29 RyRs [8, 16,21{23] and are also known to exhibit increased expression in pathological 30 conditions [24], here we use computational modelling to test a hypothesis that IP3R 31 2+ 2+ 2+ Ca release modulates Ca spark activity, and thereby affects cytosolic Ca and 32 ECC. 33 Previously, stochastic and deterministic models have been developed which 34 2+ investigate the properties of RyRs in generating Ca sparks [18,25{30]. Computational 35 models of IP3Rs have also been developed primarily for various cell types where IP3Rs 36 2+ are the predominant intracellular Ca release channel [31{35]. Deterministic temporal 37 August 13, 2020 2/17 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.13.249144; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Fig 1. Schematic illustrations of the key components at the dyadic junction underlying calcium sparks: (A) elements of Ca2+ signalling in a dyad and (B) illustration of an average Ca2+ spark. models have been proposed that include both IP3Rs and RyRs, but do not provide 38 2+ information about sparks or spatial interactions between these two SR Ca release 39 2+ channels [36, 37]. To date, the role of IP3Rs in Ca spark formation, including both 40 RyR and IP3Rs in a stochastic computational model, has not been investigated. 41 In this study, we investigate the influence that IP3R activation may have in the 42 2+ shape and temporal behavior of Ca sparks through stochastic interaction between 43 IP3Rs and RyRs. We creates a 1D spatial model of cardiomyocyte dyads containing 44 2+ stochastically opening RyR and IP3Rs. Using this model, we examine how Ca 45 -mediated interaction (crosstalk) between IP3R and RyR channels impacts the 46 2+ spatio-temporal profile of the Ca spark. We investigate the sensitivity of spark 47 initiation and shape to a range of IP3R gating parameters and IP3 concentration. Our 48 2+ findings suggest that for low IP3 concentrations, Ca release via IP3Rs is insufficient 49 to initiate sparks but they increase the probability of spark events without changing 50 2+ spark shape. The model also suggests that a small sustained Ca flux from active 51 IP3Rs diffusing to neighbouring RyRs can trigger spark formation. 52 Materials and methods 53 Model formulation 54 We model the transport of Ca2+ as a reaction-diffusion system that describes the 2+ movement of Ca in three compartments: cytosolic (Cac); junctional sarcoplasmic reticulum (CaJSR); and network sarcoplasmic reticulum (CaNSR): @[Ca ] c = D r2[Ca ] + J − J + J (1) @t c c buff SERCA release @[Ca ] JSR = β (−J + J ) (2) @t JSR release refill @[Ca ] NSR = D r2[Ca ] + J − J ; (3) @t NSR NSR SERCA refill 2+ where Dc and DNSR represent the Ca diffusivities in the cytosol and the network SR 55 respectively (the junctional SR is assumed to be a small and hence well-mixed volume).