BondGraphs.jl: composable energy-based modelling in systems biology.

SUMMARY: BondGraphs.jl is a Julia implementation of bond graphs. Bond graphs provide a modelling framework that describes energy flow through a physical system and by construction enforce thermodynamic constraints. The framework is widely used in engineering and has recently been shown to be a powerful approach for modelling biology. Models are mutable, hierarchical, multiscale, and multiphysics, and BondGraphs.jl is compatible with the Julia modelling ecosystem. AVAILABILITY AND IMPLEMENTATION: BondGraphs.jl is freely available under the MIT license. Source code and documentation can be found at https://github.com/jedforrest/BondGraphs.jl.

InsP3R-RyR channel crosstalk augments sarcoplasmic reticulum Ca2+ release and arrhythmogenic activity in post-MI pig cardiomyocytes.

Ca2+ transients (CaT) underlying cardiomyocyte (CM) contraction require efficient Ca2+ coupling between sarcolemmal Ca2+ channels and sarcoplasmic reticulum (SR) ryanodine receptor Ca2+ channels (RyR) for their generation; reduced coupling in disease contributes to diminished CaT and arrhythmogenic Ca2+ events. SR Ca2+ release also occurs via inositol 1,4,5-trisphosphate receptors (InsP3R) in CM. While this pathway contributes negligeably to Ca2+ handling in healthy CM, rodent studies support a role in altered Ca2+ dynamics and arrhythmogenic Ca2+ release involving InsP3R crosstalk with RyRs in disease. Whether this mechanism persists in larger mammals with lower T-tubular density and coupling of RyRs is not fully resolved. We have recently shown an arrhythmogenic action of InsP3-induced Ca2+ release (IICR) in end stage human heart failure (HF), often associated with underlying ischemic heart disease (IHD). How IICR contributes to early stages of disease is however not determined but highly relevant. To access this stage, we chose a porcine model of IHD, which shows substantial remodelling of the area adjacent to the infarct. In cells from this region, IICR preferentially augmented Ca2+ release from non-coupled RyR clusters that otherwise showed delayed activation during the CaT. IICR in turn synchronised Ca2+ release during the CaT but also induced arrhythmogenic delayed afterdepolarizations and action potentials. Nanoscale imaging identified co-clustering of InsP3Rs and RyRs, thereby allowing Ca2+-mediated channel crosstalk. Mathematical modelling supported and further delineated this mechanism of enhanced InsP3R-RyRs coupling in MI. Our findings highlight the role of InsP3R-RyR channel crosstalk in Ca2+ release and arrhythmia during post-MI remodelling.

Role of actin filaments and cis binding in cadherin clustering and patterning.

Cadherins build up clusters to maintain intercellular contact through trans and cis (lateral) bindings. Meanwhile, interactions between cadherin and the actin cytoskeleton through cadherin/F-actin linkers can affect cadherin dynamics by corralling and tethering cadherin molecules locally. Despite many experimental studies, a quantitative, mechanistic understanding of how cadherin and actin cytoskeleton interactions regulate cadherin clustering does not exist. To address this gap in knowledge, we developed a coarse-grained computational model of cadherin dynamics and their interaction with the actin cortex underlying the cell membrane. Our simulation predictions suggest that weak cis binding affinity between cadherin molecules can facilitate large cluster formation. We also found that cadherin movement inhibition by actin corralling is dependent on the concentration and length of actin filaments. This results in changes in cadherin clustering behaviors, as reflected by differences in cluster size and distribution as well as cadherin monomer trajectory. Strong cadherin/actin binding can enhance trans and cis interactions as well as cadherin clustering. By contrast, with weak cadherin/actin binding affinity, a competition between cadherin-actin binding and cis binding for a limited cadherin pool leads to temporary and unstable cadherin clusters.

Cortical tension initiates the positive feedback loop between cadherin and F-actin.

Adherens junctions physically link two cells at their contact interface via extracellular binding between cadherin molecules and intracellular interactions between cadherins and the actin cytoskeleton. Cadherin and actomyosin cytoskeletal dynamics are regulated reciprocally by mechanical and chemical signals, which subsequently determine the strength of cell-cell adhesions and the emergent organization and stiffness of the tissues they form. However, an understanding of the integrated system is lacking. We present a new mechanistic computational model of intercellular junction maturation in a cell doublet to investigate the mechanochemical cross talk that regulates adherens junction formation and homeostasis. The model couples a two-dimensional lattice-based simulation of cadherin dynamics with a reaction-diffusion representation of the reorganising actomyosin network through its regulation by Rho signalling at the intracellular junction. We demonstrate that local immobilization of cadherin induces cluster formation in a cis-less-dependent manner. We then recapitulate the process of cell-cell contact formation. Our model suggests that cortical tension applied on the contact rim can explain the ring distribution of cadherin and actin filaments (F-actin) on the cell-cell contact of the cell doublet. Furthermore, we propose and test the hypothesis that cadherin and F-actin interact like a positive feedback loop, which is necessary for formation of the ring structure. Different patterns of cadherin distribution were observed as an emergent property of disturbances of this positive feedback loop. We discuss these findings in light of available experimental observations on underlying mechanisms related to cadherin/F-actin binding and the mechanical environment.

EM-stellar: benchmarking deep learning for electron microscopy image segmentation.

MOTIVATION: The inherent low contrast of electron microscopy (EM) datasets presents a significant challenge for rapid segmentation of cellular ultrastructures from EM data. This challenge is particularly prominent when working with high-resolution big-datasets that are now acquired using electron tomography and serial block-face imaging techniques. Deep learning (DL) methods offer an exciting opportunity to automate the segmentation process by learning from manual annotations of a small sample of EM data. While many DL methods are being rapidly adopted to segment EM data no benchmark analysis has been conducted on these methods to date. RESULTS: We present EM-stellar, a platform that is hosted on Google Colab that can be used to benchmark the performance of a range of state-of-the-art DL methods on user-provided datasets. Using EM-stellar we show that the performance of any DL method is dependent on the properties of the images being segmented. It also follows that no single DL method performs consistently across all performance evaluation metrics. AVAILABILITY AND IMPLEMENTATION: EM-stellar (code and data) is written in Python and is freely available under MIT license on GitHub (https://github.com/cellsmb/em-stellar). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Surface area-to-volume ratio, not cellular viscoelasticity, is the major determinant of red blood cell traversal through small channels.

The remarkable deformability of red blood cells (RBCs) depends on the viscoelasticity of the plasma membrane and cell contents and the surface area to volume (SA:V) ratio; however, it remains unclear which of these factors is the key determinant for passage through small capillaries. We used a microfluidic device to examine the traversal of normal, stiffened, swollen, parasitised and immature RBCs. We show that dramatic stiffening of RBCs had no measurable effect on their ability to traverse small channels. By contrast, a moderate decrease in the SA:V ratio had a marked effect on the equivalent cylinder diameter that is traversable by RBCs of similar cellular viscoelasticity. We developed a finite element model that provides a coherent rationale for the experimental observations, based on the nonlinear mechanical behaviour of the RBC membrane skeleton. We conclude that the SA:V ratio should be given more prominence in studies of RBC pathologies.

Respiration mask waveguide optimisation for maximised speech intelligibility.

Both the scarcity and environmental impact of disposable face masks, as in the COVID-19 pandemic, have instigated the recent development of reusable masks. Such face masks reduce transmission of infectious agents and particulates, but often impact a user's ability to be understood when materials, such as silicone or hard polymers, are used. In this work, we present a numerical optimisation approach to optimise waveguide topology, where a waveguide is used to transmit and direct sound from the interior of the mask volume to the outside air. This approach allows acoustic energy to be maximised according to specific frequency bands, including those most relevant to human speech. We employ this method to convert a resuscitator mask, made of silicone, into respiration personal protective equipment (PPE) that maximises the speech intelligibility index (SII). We validate this approach experimentally as well, showing improved SII when using the fabricated device. Together, this design represents a unique and effective approach to utilize and adapt available apparatus to filter air while improving the ability to communicate effectively, including in healthcare settings.

Membrane Tension Can Enhance Adaptation to Maintain Polarity of Migrating Cells.

Migratory cells are known to adapt to environments that contain wide-ranging levels of chemoattractant. Although biochemical models of adaptation have been previously proposed, here, we discuss a different mechanism based on mechanosensing, in which the interaction between biochemical signaling and cell tension facilitates adaptation. We describe and analyze a model of mechanochemical-based adaptation coupling a mechanics-based physical model of cell tension coupled with the wave-pinning reaction-diffusion model for Rac GTPase activity. The mathematical analysis of this model, simulations of a simplified one-dimensional cell geometry, and two-dimensional finite element simulations of deforming cells reveal that as a cell protrudes under the influence of high stimulation levels, tension-mediated inhibition of Rac signaling causes the cell to polarize even when initially overstimulated. Specifically, tension-mediated inhibition of Rac activation, which has been experimentally observed in recent years, facilitates this adaptation by countering the high levels of environmental stimulation. These results demonstrate how tension-related mechanosensing may provide an alternative (and potentially complementary) mechanism for cell adaptation.

Ca2+ Release via IP3 Receptors Shapes the Cardiac Ca2+ Transient for Hypertrophic Signaling.

Calcium (Ca2+) plays a central role in mediating both contractile function and hypertrophic signaling in ventricular cardiomyocytes. L-type Ca2+ channels trigger release of Ca2+ from ryanodine receptors for cellular contraction, whereas signaling downstream of G-protein-coupled receptors stimulates Ca2+ release via inositol 1,4,5-trisphosphate receptors (IP3Rs), engaging hypertrophic signaling pathways. Modulation of the amplitude, duration, and duty cycle of the cytosolic Ca2+ contraction signal and spatial localization have all been proposed to encode this hypertrophic signal. Given current knowledge of IP3Rs, we develop a model describing the effect of functional interaction (cross talk) between ryanodine receptor and IP3R channels on the Ca2+ transient and examine the sensitivity of the Ca2+ transient shape to properties of IP3R activation. A key result of our study is that IP3R activation increases Ca2+ transient duration for a broad range of IP3R properties, but the effect of IP3R activation on Ca2+ transient amplitude is dependent on IP3 concentration. Furthermore we demonstrate that IP3-mediated Ca2+ release in the cytosol increases the duty cycle of the Ca2+ transient, the fraction of the cycle for which [Ca2+] is elevated, across a broad range of parameter values and IP3 concentrations. When coupled to a model of downstream transcription factor (NFAT) activation, we demonstrate that there is a high correspondence between the Ca2+ transient duty cycle and the proportion of activated NFAT in the nucleus. These findings suggest increased cytosolic Ca2+ duty cycle as a plausible mechanism for IP3-dependent hypertrophic signaling via Ca2+-sensitive transcription factors such as NFAT in ventricular cardiomyocytes.

Multimodal analysis of Plasmodium knowlesi-infected erythrocytes reveals large invaginations, swelling of the host cell, and rheological defects.

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block-face scanning electron microscopy to explore the topography of P. knowlesi-infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi-infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python-based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi-infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.

Insights on the impact of mitochondrial organisation on bioenergetics in high-resolution computational models of cardiac cell architecture.

Recent electron microscopy data have revealed that cardiac mitochondria are not arranged in crystalline columns but are organised with several mitochondria aggregated into columns of varying sizes spanning the cell cross-section. This raises the question-how does the mitochondrial arrangement affect the metabolite distributions within cardiomyocytes and what is its impact on force dynamics? Here, we address this question by employing finite element modeling of cardiac bioenergetics on computational meshes derived from electron microscope images. Our results indicate that heterogeneous mitochondrial distributions can lead to significant spatial variation across the cell in concentrations of inorganic phosphate, creatine (Cr) and creatine phosphate (PCr). However, our model predicts that sufficient activity of the creatine kinase (CK) system, coupled with rapid diffusion of Cr and PCr, maintains near uniform ATP and ADP ratios across the cell cross sections. This homogenous distribution of ATP and ADP should also evenly distribute force production and twitch duration with contraction. These results suggest that the PCr shuttle and associated enzymatic reactions act to maintain uniform force dynamics in the cell despite the heterogeneous mitochondrial organization. However, our model also predicts that under hypoxia activity of mitochondrial CK enzymes and diffusion of high-energy phosphate compounds may be insufficient to sustain uniform ATP/ADP distribution and hence force generation.

Assessment of single beat end-systolic elastance methods for quantifying ventricular contractility.

Multi-beat end-systolic elastance (EMB) is considered a gold-standard index of ventricular contractility. However, it is difficult to measure clinically due to the need for transient manipulation of ventricular preload or afterload. We compared the performance of 5 'single-beat' methods that do not require loading interventions, for estimating the equivalent of EMB. In 7 sheep instrumented with a micromanometer/conductance catheter, single-beat methods were compared with EMB, obtained after transiently decreasing preload or increasing afterload under a broad range of heart rates and inotropic conditions. The single-beat elastance (ESB) method described by Shishido et al. (Circulation 102(16):1983-1989, 2000) had the highest correlation (R = 0.69, y = 0.52x + 0.43) with EMB, although the absolute accuracy was poor. Interestingly, for all methods tested, a higher correlation was observed when EMB was obtained with an afterload increase (R = 0.47 - 0.78) rather than a preload reduction (R = 0.07-0.57). Within-animal regression coefficients were higher than those obtained from pooled data, with excellent within-animal correlation observed for Shishido et al. method (0.73 ≤ R ≤ 0.96) when using afterload increase as the loading intervention. We conclude that (1) current methods perform better when using an afterload increase to obtain reference EMB, (2) intra-individual ESB comparisons may be more reliable than inter-individual comparisons and (3) Shishido et al.'s method demonstrated the strongest correlation with EMB. Current ESB methods have limited and variable accuracy, but may hold promise for tracking relative changes in ventricular contractility in individuals.

Automated segmentation of cardiomyocyte Z-disks from high-throughput scanning electron microscopy data.

BACKGROUND: With the advent of new high-throughput electron microscopy techniques such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion-beam scanning electron microscopy (FIB-SEM) biomedical scientists can study sub-cellular structural mechanisms of heart disease at high resolution and high volume. Among several key components that determine healthy contractile function in cardiomyocytes are Z-disks or Z-lines, which are located at the lateral borders of the sarcomere, the fundamental unit of striated muscle. Z-disks play the important role of anchoring contractile proteins within the cell that make the heartbeat. Changes to their organization can affect the force with which the cardiomyocyte contracts and may also affect signaling pathways that regulate cardiomyocyte health and function. Compared to other components in the cell, such as mitochondria, Z-disks appear as very thin linear structures in microscopy data with limited difference in contrast to the remaining components of the cell. METHODS: In this paper, we propose to generate a 3D model of Z-disks within single adult cardiac cells from an automated segmentation of a large serial-block-face scanning electron microscopy (SBF-SEM) dataset. The proposed fully automated segmentation scheme is comprised of three main modules including "pre-processing", "segmentation" and "refinement". We represent a simple, yet effective model to perform segmentation and refinement steps. Contrast stretching, and Gaussian kernels are used to pre-process the dataset, and well-known "Sobel operators" are used in the segmentation module. RESULTS: We have validated our model by comparing segmentation results with ground-truth annotated Z-disks in terms of pixel-wise accuracy. The results show that our model correctly detects Z-disks with 90.56% accuracy. We also compare and contrast the accuracy of the proposed algorithm in segmenting a FIB-SEM dataset against the accuracy of segmentations from a machine learning program called Ilastik and discuss the advantages and disadvantages that these two approaches have. CONCLUSIONS: Our validation results demonstrate the robustness and reliability of our algorithm and model both in terms of validation metrics and in terms of a comparison with a 3D visualisation of Z-disks obtained using immunofluorescence based confocal imaging.

An automated workflow for segmenting single adult cardiac cells from large-volume serial block-face scanning electron microscopy data.

This paper presents a new algorithm to automatically segment the myofibrils, mitochondria and nuclei within single adult cardiac cells that are part of a large serial-block-face scanning electron microscopy (SBF-SEM) dataset. The algorithm only requires a set of manually drawn contours that roughly demarcate the cell boundary at routine slice intervals (every 50th, for example). The algorithm correctly classified pixels within the single cell with 97% accuracy when compared to manual segmentations. One entire cell and the partial volumes of two cells were segmented. Analysis of segmentations within these cells showed that myofibrils and mitochondria occupied 47.5% and 51.6% on average respectively, while the nuclei occupy 0.7% of the cell for which the entire volume was captured in the SBF-SEM dataset. Mitochondria clustering increased at the periphery of the nucleus region and branching points of the cardiac cell. The segmentations also showed high area fraction of mitochondria (up to 70% of the 2D image slice) in the sub-sarcolemmal region, whilst it was closer to 50% in the intermyofibrillar space. We finally demonstrate that our segmentations can be turned into 3D finite element meshes for cardiac cell computational physiology studies. We offer our large dataset and MATLAB implementation of the algorithm for research use at www.github.com/CellSMB/sbfsem-cardiac-cell-segmenter/. We anticipate that this timely tool will be of use to cardiac computational and experimental physiologists alike who study cardiac ultrastructure and its role in heart function.

Changes in mitochondrial morphology and organization can enhance energy supply from mitochondrial oxidative phosphorylation in diabetic cardiomyopathy.

Diabetic cardiomyopathy is accompanied by metabolic and ultrastructural alterations, but the impact of the structural changes on metabolism itself is yet to be determined. Morphometric analysis of mitochondrial shape and spatial organization within transverse sections of cardiomyocytes from control and streptozotocin-induced type I diabetic Sprague-Dawley rats revealed that mitochondria are 20% smaller in size while their spatial density increases by 53% in diabetic cells relative to control myocytes. Diabetic cells formed larger clusters of mitochondria (60% more mitochondria per cluster) and the effective surface-to-volume ratio of these clusters increased by 22.5%. Using a biophysical computational model we found that this increase can have a moderate compensatory effect by increasing the availability of ATP in the cytosol when ATP synthesis within the mitochondrial matrix is compromised.

Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes.

Spatio-temporal dynamics of intracellular calcium, [Ca2+]i, regulate the contractile function of cardiac muscle cells. Measuring [Ca2+]i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca2+]i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca2+ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca2+]i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca2+]i transient and of the simulated fluorescence intensity signal, F/F0, reached values similar to that found in the literature ([Ca2+]i ≈1 µM; F/F0≈5.5). However, our model predicted the variation in [Ca2+]i to be between 0.3 and 12.7 µM (~3 to 100 fold from resting value of 0.1 µM) and the corresponding F/F0 signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca2+]i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca2+]i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca2+]i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.

Critical review of single-cell mechanotyping approaches for biomedical applications.

Accurate mechanical measurements of cells has the potential to improve diagnostics, therapeutics and advance understanding of disease mechanisms, where high-resolution mechanical information can be measured by deforming individual cells. Here we evaluate recently developed techniques for measuring cell-scale stiffness properties; while many such techniques have been developed, much of the work examining single-cell stiffness is impacted by difficulties in standardization and comparability, giving rise to large variations in reported mechanical moduli. We highlight the role of underlying mechanical theories driving this variability, and note opportunities to develop novel mechanotyping devices and theoretical models that facilitate convenient and accurate mechanical characterisation. Moreover, many high-throughput approaches are confounded by factors including cell size, surface friction, natural population heterogeneity and convolution of elastic and viscous contributions to cell deformability. We nevertheless identify key approaches based on deformability cytometry as a promising direction for further development, where both high-throughput and accurate single-cell resolutions can be realized.

Genome-wide identification of replication fork stalling/pausing sites and the interplay between RNA Pol II transcription and DNA replication progression.

BACKGROUND: DNA replication progression can be affected by the presence of physical barriers like the RNA polymerases, leading to replication stress and DNA damage. Nonetheless, we do not know how transcription influences overall DNA replication progression. RESULTS: To characterize sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints during S-phase to identify replication fork/stalling hotspots as replication progresses through the genome. These sites are typically associated with increased DNA damage, overlapped with fragile sites and with breakpoints of rearrangements identified in cancers but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA polymerase II transcription, we find that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. Indeed, we find that slowing down transcription elongation slows down directly replication progression through genes. This indicates that transcription and replication can coexist over the same regions. Importantly, rearrangements found in cancers overlapping transcription-replication collision sites are detected in non-transformed cells and increase following treatment with ATM and ATR inhibitors. At the same time, we find instances where transcription activity favors replication progression because it reduces histone density. CONCLUSIONS: Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability by the coexistence of these two processes.

Sub-wavelength acoustic stencil for tailored micropatterning.

Acoustofluidic devices are ideal for biomedical micromanipulation applications, with high biocompatibility and the ability to generate force gradients down to the scale of cells. However, complex and designed patterning at the microscale remains challenging. In this work we report an acoustofluidic approach to direct particles and cells within a structured surface in arbitrary configurations. Wells, trenches and cavities are embedded in this surface. Combined with a half-wavelength acoustic field, together these form an 'acoustic stencil' where arbitrary cell and particle arrangements can be reversibly generated. Here a bulk-wavemode lithium niobate resonator generates multiplexed parallel patterning via a multilayer resonant geometry, where cell-scale resolution is accomplished via structured sub-wavelength microfeatures. Uniquely, this permits simultaneous manipulation in a unidirectional, device-spanning single-node field across scalable ∼cm2 areas in a microfluidic device. This approach is demonstrated via patterning of 5, 10 and 15 µm particles and 293-F cells in a variety of arrangements, where these activities are enabling for a range of cell studies and tissue engineering applications via the generation of highly complex and designed acoustic patterns at the microscale.

IP3R activity increases propensity of RyR-mediated sparks by elevating dyadic [Ca2＋].

Calcium (Ca2＋) plays a critical role in the excitation contraction coupling (ECC) process that mediates the contraction of cardiomyocytes during each heartbeat. While ryanodine receptors (RyRs) are the primary Ca2＋ channels responsible for generating the cell-wide Ca2＋ transients during ECC, Ca2＋ release, via inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are also reported in cardiomyocytes to elicit ECC-modulating effects. Recent studies suggest that the localization of IP3Rs at dyads grant their ability to modify the occurrence of Ca2＋ sparks (elementary Ca2＋ release events that constitute cell wide Ca2＋ releases associated with ECC) which may underlie their modulatory influence on ECC. Here, we aim to uncover the mechanism by which dyad-localized IP3Rs influence Ca2＋ spark dynamics. To this end, we developed a mathematical model of the dyad that incorporates the behaviour of IP3Rs, in addition to RyRs, to reveal the impact of their activity on local Ca2＋ handling and consequent Ca2＋ spark occurrence and its properties. Consistent with published experimental data, our model predicts that the propensity for Ca2＋ spark formation increases in the presence of IP3R activity. Our simulations support the hypothesis that IP3Rs elevate Ca2＋ in the dyad, sensitizing proximal RyRs towards activation and hence Ca2＋ spark formation. The stochasticity of IP3R gating is an important aspect of this mechanism. However, dyadic IP3R activity lowers the Ca2＋ available in the junctional sarcoplasmic reticulum (JSR) for release, thus resulting in Ca2＋ sparks with similar durations but lower amplitudes.