Isolation and reconstruction of cardiac mitochondria from SBEM images using a deep learning-based method.

Mitochondrial morphological defects are a common feature of diseased cardiac myocytes. However, quantitative assessment of mitochondrial morphology is limited by the time-consuming manual segmentation of electron micrograph (EM) images. To advance understanding of the relation between morphological defects and dysfunction, an efficient morphological reconstruction method is desired to enable isolation and reconstruction of mitochondria from EM images. We propose a new method for isolating and reconstructing single mitochondria from serial block-face scanning EM (SBEM) images. CDeep3M, a cloud-based deep learning network for EM images, was used to segment mitochondrial interior volumes and boundaries. Post-processing was performed using both the predicted interior volume and exterior boundary to isolate and reconstruct individual mitochondria. Series of SBEM images from two separate cardiac myocytes were processed. The highest F1-score was 95% using 50 training datasets, greater than that for previously reported automated methods and comparable to manual segmentations. Accuracy of separation of individual mitochondria was 80% on a pixel basis. A total of 2315 mitochondria in the two series of SBEM images were evaluated with a mean volume of 0.78 µm3. The volume distribution was very broad and skewed; the most frequent mitochondria were 0.04-0.06 µm3, but mitochondria larger than 2.0 µm3 accounted for more than 10% of the total number. The average short-axis length was 0.47 µm. Primarily longitudinal mitochondria (0-30 degrees) were dominant (54%). This new automated segmentation and separation method can help quantitate mitochondrial morphology and improve understanding of myocyte structure-function relationships.

An integrated finite element simulation of cardiomyocyte function based on triphasic theory.

In numerical simulations of cardiac excitation-contraction coupling, the intracellular potential distribution and mobility of cytosol and ions have been mostly ignored. Although the intracellular potential gradient is small, during depolarization it can be a significant driving force for ion movement, and is comparable to diffusion in terms of net flux. Furthermore, fluid in the t-tubules is thought to advect ions to facilitate their exchange with the extracellular space. We extend our previous finite element model that was based on triphasic theory to examine the significance of these factors in cardiac physiology. Triphasic theory allows us to study the behavior of solids (proteins), fluids (cytosol) and ions governed by mechanics and electrochemistry in detailed subcellular structures, including myofibrils, mitochondria, the sarcoplasmic reticulum, membranes, and t-tubules. Our simulation results predicted an electrical potential gradient inside the t-tubules at the onset of depolarization, which corresponded to the Na(+) channel distribution therein. Ejection and suction of fluid between the t-tubules and the extracellular compartment during isometric contraction were observed. We also examined the influence of t-tubule morphology and mitochondrial location on the electrophysiology and mechanics of the cardiomyocyte. Our results confirm that the t-tubule structure is important for synchrony of Ca(2+) release, and suggest that mitochondria in the sub-sarcolemmal region might serve to cancel Ca(2+) inflow through surface sarcolemma, thereby maintaining the intracellular Ca(2+) environment in equilibrium.

Distinct functional roles of cardiac mitochondrial subpopulations revealed by a 3D simulation model.

Experimental characterization of two cardiac mitochondrial subpopulations, namely, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), has been hampered by technical difficulties, and an alternative approach is eagerly awaited. We previously developed a three-dimensional computational cardiomyocyte model that integrates electrophysiology, metabolism, and mechanics with subcellular structure. In this study, we further developed our model to include intracellular oxygen diffusion, and determined whether mitochondrial localization or intrinsic properties cause functional variations. For this purpose, we created two models: one with equal SSM and IFM properties and one with IFM having higher activity levels. Using these two models to compare the SSM and IFM responses of [Ca(2+)], tricarboxylic acid cycle activity, [NADH], and mitochondrial inner membrane potential to abrupt changes in pacing frequency (0.25-2 Hz), we found that the reported functional differences between these subpopulations appear to be mostly related to local [Ca(2+)] heterogeneity, and variations in intrinsic properties only serve to augment these differences. We also examined the effect of hypoxia on mitochondrial function. Under normoxic conditions, intracellular oxygen is much higher throughout the cell than the half-saturation concentration for oxidative phosphorylation. However, under limited oxygen supply, oxygen is mostly exhausted in SSM, leaving the core region in an anoxic condition. Reflecting this heterogeneous oxygen environment, the inner membrane potential continues to decrease in IFM, whereas it is maintained to nearly normal levels in SSM, thereby ensuring ATP supply to this region. Our simulation results provide clues to understanding the origin of functional variations in two cardiac mitochondrial subpopulations and their differential roles in maintaining cardiomyocyte function as a whole.

Mitochondrial colocalization with Ca2+ release sites is crucial to cardiac metabolism.

In cardiomyocyte subcellular structures, colocalization of mitochondria with Ca2+ release sites is implicated in regulation of cardiac energetics by facilitating Ca2+ influx into mitochondria to modulate the tricarboxylic acid (TCA) cycle. However, current experimental techniques limit detailed examination of this regulatory mechanism. Earlier, we developed a three-dimensional (3D) finite-element cardiomyocyte model featuring a subcellular structure that integrates excitation-contraction coupling and energy metabolism. Here, using this model, we examined the influence of distance between mitochondria and Ca2+ release sites by comparing a normal (50-nm) distance model and a large (200-nm) distance model (LD). The influence of distance was minimal under a low pacing rate (0.25 Hz), but under a higher pacing rate (2 Hz), lower levels of mitochondrial Ca2+ and NADH, elevated phosphate, and suppressed force generation became apparent in the LD model. Such differences became greater when functional impairments (reduced TCA cycle activity, uncoupling effect, and failing excitation-contraction coupling) were additionally imposed. We concluded that juxtaposition of the mitochondria and the Ca2+ release sites is crucial for rapid signal transmission to maintain cardiac-energy balance. The idealized 3D model of cardiac excitation-contraction and metabolism is a powerful tool to study cardiac energetics.

Multi-scale simulations of cardiac electrophysiology and mechanics using the University of Tokyo heart simulator.

The importance and need for an integrative mathematical modeling approach in the biological and medical fields is currently well recognized. Such an approach is crucial in understanding the complexity of hierarchical biological systems increasingly revealed by active researches in molecular and cellular biology. Particularly in cardiac functioning, modeling must cover such diverse phenomena as solid mechanics, fluid dynamics, electricity and biochemistry. Recent advancements in computational science and the development of high-performance computers have enabled the creation of multi-scale, multi-physics simulation heart models using the finite element method. Although whole heart or ventricular models of electrophysiology involving electro-mechanics with or without blood flow dynamics have been reported, to our knowledge no single model has yet succeeded in completely reproducing the behavior of the heart from the subcellular to whole organ levels. In this article, we present a brief methodology-focused review on some of the essential components for multi-scale, multi-physics heart modeling. A perspective of heart modeling in the era of high performance computing is also presented.

Critical role of cardiac t-tubule system for the maintenance of contractile function revealed by a 3D integrated model of cardiomyocytes.

T-tubules in mammalian ventricular myocytes constitute an elaborate system for coupling membrane depolarization with intracellular Ca(2+) signaling to control cardiac contraction. Deletion of t-tubules (detubulation) has been reported in heart diseases, although the complex nature of the cardiac excitation-contraction (E-C) coupling process makes it difficult to experimentally establish causal relationships between detubulation and cardiac dysfunction. Alternatively, numerical simulations incorporating the t-tubule system have been proposed to elucidate its functional role. However, the majority of models treat the subcellular spaces as lumped compartments, and are thus unable to dissect the impact of morphological changes in t-tubules. We developed a 3D finite element model of cardiomyocytes in which subcellular components including t-tubules, myofibrils, sarcoplasmic reticulum, and mitochondria were modeled and realistically arranged. Based on this framework, physiological E-C coupling was simulated by simultaneously solving the reaction-diffusion equation and the mechanical equilibrium for the mathematical models of electrophysiology and contraction distributed among these subcellular components. We then examined the effect of detubulation in this model by comparing with and without the t-tubule system. This model reproduced the Ca(2+) transients and contraction observed in experimental studies, including the response to beta-adrenergic stimulation, and provided detailed information beyond the limits of experimental approaches. In particular, the analysis of sarcomere dynamics revealed that the asynchronous contraction caused by a large detubulated region can lead to impairment of myocyte contractile efficiency. These data clearly demonstrate the importance of the t-tubule system for the maintenance of contractile function.

A three-dimensional simulation model of cardiomyocyte integrating excitation-contraction coupling and metabolism.

Recent studies have revealed that Ca(2+) not only regulates the contraction of cardiomyocytes, but can also function as a signaling agent to stimulate ATP production by the mitochondria. However, the spatiotemporal resolution of current experimental techniques limits our investigative capacity to understand this phenomenon. Here, we created a detailed three-dimensional (3D) cardiomyocyte model to study the subcellular regulatory mechanisms of myocardial energetics. The 3D cardiomyocyte model was based on the finite-element method, with detailed subcellular structures reproduced, and it included all elementary processes involved in cardiomyocyte electrophysiology, contraction, and ATP metabolism localized to specific loci. The simulation results were found to be reproducible and consistent with experimental data regarding the spatiotemporal pattern of cytosolic, intrasarcoplasmic-reticulum, and mitochondrial changes in Ca(2+); as well as changes in metabolite levels. Detailed analysis suggested that although the observed large cytosolic Ca(2+) gradient facilitated uptake by the mitochondrial Ca(2+) uniporter to produce cyclic changes in mitochondrial Ca(2+) near the Z-line region, the average mitochondrial Ca(2+) changes slowly. We also confirmed the importance of the creatine phosphate shuttle in cardiac energy regulation. In summary, our 3D model provides a powerful tool for the study of cardiac function by overcoming some of the spatiotemporal limitations of current experimental approaches.