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Calcium (Ca 2+ ) plays a central role in mediating both contractile function and hypertrophic signalling in ventricular cardiomyocytes. L-type Ca 2+ channels trigger release of Ca 2+ from ryanodine receptors (RyRs) for cellular contraction, while signalling downstream of Gq coupled receptors stimulates Ca 2+ release via inositol 1,4,5-trisphosphate receptors (IP 3 Rs), engaging hypertrophic signalling pathways. Modulation of the amplitude, duration, and duty cycle of the cytosolic Ca 2+ contraction signal, and spatial localisation, have all been proposed to encode this hypertrophic signal. Given current knowledge of IP 3 Rs, we develop a model describing the effect of functional interaction (cross-talk) between RyR and IP 3 R channels on the Ca 2+ transient, and examine the sensitivity of the Ca 2+ transient shape to properties of IP 3 R activation. A key result of our study is that IP 3 R activation increases Ca 2+ transient duration for a broad range of IP 3 R properties, but the effect of IP 3 R activation on Ca 2+ transient amplitude is dependent on IP 3 concentration. Furthermore we demonstrate that IP 3 -mediated Ca 2+ release in the cytosol increases the duty cycle of the Ca 2+ transient, the fraction of the cycle for which [Ca 2+ ] is elevated, across a broad range of parameter values and IP 3 concentrations. When coupled to a model of downstream transcription factor (NFAT) activation, we demonstrate that there is a high correspondence between the Ca 2+ transient duty cycle and the proportion of activated NFAT in the nucleus. These findings suggest increased cytosolic Ca 2+ duty cycle as a plausible mechanism for IP 3 -dependent hypertrophic signalling via Ca 2+ -sensitive transcription factors such as NFAT in ventricular cardiomyocytes. SIGNIFICANCE Many studies have identified a role for IP 3 R-mediated Ca 2+ signalling in cardiac hypertrophy, however the mechanism by which this signal is communicated within the cardiomyocyte remains unclear. We present a mathematical model of functional interactions between RyR and IP 3 R channels. We show that IP 3 -mediated Ca 2+ release is capable of providing a modest increase to the duty cycle of the calcium signal, which has been shown experimentally to lead to NFAT activation, and hence hypertrophic signalling. Through a parameter sensitivity analysis we demonstrate that the duty cycle is increased with IP 3 over a broad parameter regime, indicating that this mechanism is robust, and we show that an increase in Ca 2+ duty cycle increases nuclear NFAT activation. These findings suggest a plausible mechanism for IP3R-dependent hypertrophic signalling in cardiomyocytes. INTRODUCTIONCalcium is a universal second messenger that plays a role in controlling many cellular processes across a wide variety of cell types; ranging from fertilisation, cell contraction, and cell growth, to cell death (1, 2). Precisely how Ca 2+ fulfills each of these † These authors contributed equally to the supervision of this work.

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

Companion cells and sieve elements play an essential role in vascular plants and yet the details of the metabolism that underpins their function remain largely unknown. Here we construct a tissue-scale flux balance analysis (FBA) model to describe the metabolism of phloem loading in a mature Arabidopsis (Arabidopsis thaliana) leaf. We explore the potential metabolic interactions between mesophyll cells, companion cells, and sieve elements based on the current understanding of the physiology of phloem tissue and through the use of cell-type-specific transcriptome data as a weighting in our model. We find that companion cell chloroplasts likely play a very different role to mesophyll chloroplasts. Our model suggests that, rather than carbon capture, the most crucial function of companion cell chloroplasts is to provide photosynthetically-generated ATP to the cytosol. Additionally, our model predicts that the metabolites imported into the companion cell are not necessarily the same metabolites that are exported in phloem sap; phloem loading is more efficient if certain amino acids are synthesised in the phloem tissue. Surprisingly, in our model predictions the proton pumping pyrophosphatase (H + -PPiase) is a more efficient contributor to the energisation of the companion cell plasma membrane than the H + -ATPase. A computational model provides insight into the metabolism behind Arabidopsis phloem loading, predicting a crucial role for companion cell chloroplasts in phloem loading energy metabolism. kiad154_Supplementary_Data.zip

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 determine the mechanism by which IP3Rs modify Ca2+ 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 that Ca2+ release via IP3Rs result in increased propensity for spark initiation within the cardiac dyad. Our simulations suggest that opening of IP3Rs elevates Ca2+ 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 IP3R play a critical role in modulating Ca2+ signaling for excitation contraction coupling.