Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

University of Melbourne

A major motivation for the use of super-resolution imaging methods in the investigation of cardiac biophysics has been the insight from biophysical considerations and detailed mathematical modeling that the spatial structure and protein organisation at the scale of nanometres can have enormous implications for calcium signalling in cardiac muscle. We illustrate the use of dSTORM based super-resolution in optically thick (∼10 μm) tissue slices of rat ventricular tissue to visualize proteins at the cardiac Z-disk and compare those images with confocal (diffraction-limited) as well as electron microscopy (EM) data which still provides a benchmark in terms of resolution. α-actinin is an abundant protein target that effectively defines the Z-disk in striated muscle and provides a reference structure for other proteins at the Z-line and the transverse tubules. Using super-resolution imaging α-actinin labelling provides very detailed outlines of the contractile machinery which we have used to study the properties of Z-disks and the distribution of α-actinin itself. We determined the local diameters of the myo-fibrillar and non-myofibrillar space using α-actinin labelling. Comparison between confocal and super-resolution based myofibrillar masks suggested that super-resolution data was able to segment myofibrils accurately while confocal approaches were not always able to distinguish neighbouring myofibrillar bundles which resulted in overestimated diameters. The increased resolution of super-resolution methods provides qualitatively new information to improve our understanding of cardiac biophysics. Nevertheless, conventional diffraction-limited imaging still has an important role to play which we illustrate with correlative confocal and super-resolution data. 

Super-resolution fluorescence imaging to study cardiac biophysics: α-actinin distribution and Z-disk topologies in optically thick cardiac tissue slices.

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. 

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

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.

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

Cellular cytoskeletal mechanics plays a major role in many aspects of human health from organ development to wound healing, tissue homeostasis and cancer metastasis. We summarize the state-of-the-art techniques for mathematically modeling cellular stiffness and mechanics and the cytoskeletal components and factors that regulate them. We highlight key experiments that have assisted model parameterization and compare the advantages of different models that have been used to recapitulate these experiments. An overview of feed-forward mechanisms from signaling to cytoskeleton remodeling is provided, followed by a discussion of the rapidly growing niche of encapsulating feedback mechanisms from cytoskeletal and cell mechanics to signaling. We discuss broad areas of advancement that could accelerate research and understanding of cellular mechanobiology. A precise understanding of the molecular mechanisms that affect cell and tissue mechanics and function will underpin innovations in medical device technologies of the future. WIREs Syst Biol Med 2018, 10:e1407. doi: 10.1002/wsbm.1407 This article is categorized under: Models of Systems Properties and Processes > Mechanistic Models Physiology > Mammalian Physiology in Health and Disease Models of Systems Properties and Processes > Cellular Models.

Computational modeling of single-cell mechanics and cytoskeletal mechanobiology.

The malaria parasite hijacks host erythrocytes to shield itself from the immune system and proliferate. Red blood cell abnormalities can provide protection from malaria by impeding parasite invasion and growth within the cell or by compromising the ability of parasites to avoid host clearance. Here, we describe 2 -ethyl--nitrosourea-induced mouse lines,  and  , containing single-point mutations in the erythrocyte membrane skeleton gene, β spectrin (), which exhibit microcytosis but retain a relatively normal ratio of erythrocyte surface area to volume and are highly resistant to rodent malaria. We propose the major factor responsible for malaria protection is the specific clearance of mutant erythrocytes, although an enhanced clearance of uninfected mutant erythrocytes was also observed (ie, the bystander effect). Using an in vivo erythrocyte tracking assay, we established that this phenomenon occurs irrespective of host environment, precluding the involvement of nonerythrocytic cells in the resistance mechanism. Furthermore, we recapitulated this phenotype by disrupting the interaction between ankyrin-1 and β spectrin in vivo using CRISPR/Cas9 genome editing technology, thereby genetically validating a potential antimalarial target. This study sheds new light on the role of β spectrin during  infection and highlights how changes in the erythrocyte cytoskeleton can substantially influence malaria susceptibility with minimal adverse consequences for the host.

Erythrocyte β spectrin can be genetically targeted to protect mice from malaria.

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.

Vijay Rajagopal

Cell Structure and Mechanobiology Group,
Systems Biology Laboratory,
Melbourne School of Engineering,
Department of Biomedical Engineering,
Systems Biology Laboratory, Melbourne School of Engineering

University of Melbourne

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

Vijay Rajagopal

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Cell Structure and Mechanobiology Group,Systems Biology Laboratory,Melbourne School of Engineering,Department of Biomedical Engineering,Systems Biology Laboratory, Melbourne School of Engineering

University of Melbourne

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

Cell Structure and Mechanobiology Group,
Systems Biology Laboratory,
Melbourne School of Engineering,
Department of Biomedical Engineering,
Systems Biology Laboratory, Melbourne School of Engineering