Cardiac cell modelling: Observations from the heart of the cardiac physiome project

In this manuscript we review the state of cardiac cell modelling in the context of international initiatives such as the IUPS Physiome and Virtual Physiological Human Projects, which aim to integrate computational models across scales and physics. In particular we focus on the relationship between experimental data and model parameterisation across a range of model types and cellular physiological systems. Finally, in the context of parameter identification and model reuse within the Cardiac Physiome, we suggest some future priority areas for this field

EMT/MET at the crossroad of stemness, regeneration and oncogenesis. The Ying-Yang equilibrium recapitulated in cell spheroids

The epithelial-to-mesenchymal transition (EMT) is an essential trans-differentiation process, which plays a critical role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. It is the fundamental mechanism by which epithelial cells lose many of their characteristics while acquiring features typical of mesenchymal cells, such as migratory capacity and invasiveness. Depending on the contest, EMT is complemented and balanced by the reverse process, the mesenchymal-to-epithelial transition (MET). In the saving economy of the living organisms, the same (Ying-Yang) tool is integrated as a physiological strategy in embryonic development, as well as in the course of reparative or disease processes, prominently fibrosis, tumor invasion and metastasis. These mechanisms and their related signaling (e.g., TGF-β and BMPs) have been effectively studied in vitro by tissue-derived cell spheroids models. These three-dimensional (3D) cell culture systems, whose phenotype has been shown to be strongly dependent on TGF-β-regulated EMT/MET processes, present the advantage of recapitulating in vitro the hypoxic in vivo micro-environment of tissue stem cell niches and their formation. These spheroids, therefore, nicely reproduce the finely regulated Ying-Yang equilibrium, which, together with other mechanisms, can be determinant in cell fate decisions in many pathophysiological scenarios, such as differentiation, fibrosis, regeneration, and oncogenesis. In this review, current progress in the knowledge of signaling pathways affecting EMT/MET and stemness regulation will be outlined by comparing data obtained from cellular spheroids systems, as ex vivo niches of stem cells derived from normal and tumoral tissues. The mechanistic correspondence in vivo and the possible pharmacological perspective will be also explored, focusing especially on the TGF-β-related networks, as well as others, such as SNAI1, PTEN, and EGR1. This latter, in particular, for its ability to convey multiple types of stimuli into relevant changes of the cell transcriptional program, can be regarded as a heterogeneous "stress-sensor" for EMT-related inducers (growth factor, hypoxia, mechano-stress), and thus as a therapeutic target

Contractile deficits in engineered cardiac microtissues as a result of MYBPC3 deficiency and mechanical overload.

The integration of in vitro cardiac tissue models, human induced pluripotent stem cells (hiPSCs) and genome-editing tools allows for the enhanced interrogation of physiological phenotypes and recapitulation of disease pathologies. Here, using a cardiac tissue model consisting of filamentous three-dimensional matrices populated with cardiomyocytes derived from healthy wild-type (WT) hiPSCs (WT hiPSC-CMs) or isogenic hiPSCs deficient in the sarcomere protein cardiac myosin-binding protein C (MYBPC3-/- hiPSC-CMs), we show that the WT microtissues adapted to the mechanical environment with increased contraction force commensurate to matrix stiffness, whereas the MYBPC3-/- microtissues exhibited impaired force development kinetics regardless of matrix stiffness and deficient contraction force only when grown on matrices with high fibre stiffness. Under mechanical overload, the MYBPC3-/- microtissues had a higher degree of calcium transient abnormalities, and exhibited an accelerated decay of calcium dynamics as well as calcium desensitization, which accelerated when contracting against stiffer fibres. Our findings suggest that MYBPC3 deficiency and the presence of environmental stresses synergistically lead to contractile deficits in cardiac tissues

Mechano-transduction: from molecules to tissues.

External forces play complex roles in cell organization, fate, and homeostasis. Changes in these forces, or how cells respond to them, can result in abnormal embryonic development and diseases in adults. How cells sense and respond to these mechanical stimuli requires an understanding of the biophysical principles that underlie changes in protein conformation and result in alterations in the organization and function of cells and tissues. Here, we discuss mechano-transduction as it applies to protein conformation, cellular organization, and multi-cell (tissue) function

Reactive oxygen species, vascular noxs, and hypertension: focus on translational and clinical research

Significance: Reactive oxygen species (ROS) are signaling molecules that are important in physiological processes, including host defense, aging, and cellular homeostasis. Increased ROS bioavailability and altered redox signaling (oxidative stress) have been implicated in the onset and/or progression of chronic diseases, including hypertension. Recent Advances: Although oxidative stress may not be the only cause of hypertension, it amplifies blood pressure elevation in the presence of other pro-hypertensive factors, such as salt loading, activation of the renin-angiotensin-aldosterone system, and sympathetic hyperactivity, at least in experimental models. A major source for ROS in the cardiovascular-renal system is a family of nicotinamide adenine dinucleotide phosphate oxidases (Noxs), including the prototypic Nox2-based Nox, and Nox family members: Nox1, Nox4, and Nox5. Critical Issues: Although extensive experimental data support a role for increased ROS levels and altered redox signaling in the pathogenesis of hypertension, the role in clinical hypertension is unclear, as a direct causative role of ROS in blood pressure elevation has yet to be demonstrated in humans. Nevertheless, what is becoming increasingly evident is that abnormal ROS regulation and aberrant signaling through redox-sensitive pathways are important in the pathophysiological processes which is associated with vascular injury and target-organ damage in hypertension. Future Directions: There is a paucity of clinical information related to the mechanisms of oxidative stress and blood pressure elevation, and a few assays accurately measure ROS directly in patients. Such further ROS research is needed in humans and in the development of adequately validated analytical methods to accurately assess oxidative stress in the clinic

Systems Modeling to Predict Mechano-Chemo Interactions In Cardiac Fibrosis

Cardiac fibrosis poses a central challenge in preventing heart failure for patients who have suffered a cardiac injury such as myocardial infarction or aortic valve stenosis. This chronic condition is characterized by a reduction in contractile function through combined hypertrophy and excessive scar formation, and although currently prescribed therapeutics targeting hypertrophy have shown improvements in patient outcomes, pathological fibrosis remains a leading cause of reduced cardiac function for patients long-term. Cardiac fibroblasts play a key role in regulating scar formation during heart failure progression, and interacting biochemical and biomechanical cues within the myocardium guide the activation of fibroblasts and expression of extracellular matrix proteins. While targeted experimental studies of fibroblast activation have elucidated the roles of individual pathways in fibroblast activation, intracellular crosstalk between mechanotransduction and chemotransduction pathways from multiple biochemical cues has largely confounded efforts to control overall cell behavior within the myocardial environment. Computational networks of intracellular signaling can account for complex interactions between signaling pathways and provide a promising approach for identifying influential mechanisms mediating cell behavior. The overarching goal of this dissertation is to improve our understanding of complex signaling in fibroblasts by investigating the role of mechano-chemo interactions in cardiac fibroblast-mediated fibrosis using a combination of experimental studies and systems-level computational models. Firstly, using an in vitro screen of cardiac fibroblast-secreted proteins in response to combinations of biochemical stimuli and mechanical tension, we found that tension modulated cell sensitivity towards biochemical stimuli, thereby altering cell behavior based on the mechanical context. Secondly, using a curated model of fibroblast intracellular signaling, we expanded model topology to include robust mechanotransduction pathways, improved accuracy of model predictions compared to independent experimental studies, and identified mechanically dependent mechanisms-of- ction and mechano-adaptive drug candidates in a post-infarction scenario. Lastly, using an inferred network of fibroblast transcriptional regulation and model fitting to patient-specific data, we showed the utility of model-based approaches in identifying influential pathways underlying fibrotic protein expression during aortic valve stenosis and predicting patient-specific responses to pharmacological intervention. Our work suggests that computational-based approaches can generate insight into influential mechanisms among complex systems, and such tools may be promising for further therapeutic development and precision medicine

Interpretable Mechanistic and Machine Learning Models for Pre-dicting Cardiac Remodeling from Biochemical and Biomechanical Features

Biochemical and biomechanical signals drive cardiac remodeling, resulting in altered heart physiology and the precursor for several cardiac diseases, the leading cause of death for most racial groups in the USA. Reversing cardiac remodeling requires medication and device-assisted treatment such as Cardiac Resynchronization Therapy (CRT), but current interventions produce highly variable responses from patient to patient. Mechanistic modeling and Machine learning (ML) approaches have the functionality to aid diagnosis and therapy selection using various input features. Moreover, \u27Interpretable\u27 machine learning methods have helped make machine learning models fairer and more suited for clinical application. The overarching objective of this doctoral work is to develop computational models that combine an extensive array of clinically measured biochemical and biomechanical variables to enable more accurate identification of heart failure patients prone to respond positively to therapeutic interventions. In the first aim, we built an ensemble ML classification algorithm using previously acquired data from the SMART-AV CRT clinical trial. Our classification algorithm incorporated 26 patient demographic and medical history variables, 12 biomarker variables, and 18 LV functional variables, yielding correct CRT response prediction in 71% of patients. In the second aim, we employed a machine learning-based method to infer the fibrosis-related gene regulatory network from RNA-seq data from the MAGNet cohort of heart failure patients. This network identified significant interactions between transcription factors and cell synthesis outputs related to cardiac fibrosis - a critical driver of heart failure. Novel filtering methods helped us prioritize the most critical regulatory interactions of mechanistic forward simulations. In the third aim, we developed a logic-based model for the mechanistic network of cardiac fibrosis, integrating the gene regulatory network derived from aim two into a previously constructed cardiac fibrosis signaling network model. This integrated model implemented biochemical and biomechanical reactions as ordinary differential equations based on normalized Hill functions. The model elucidated the semi-quantitative behavior of cardiac fibrosis signaling complexity by capturing multi-pathway crosstalk and feedback loops. Perturbation analysis predicted the most critical nodes in the mechanistic model. Patient-specific simulations helped identify which biochemical species highly correlate with clinical measures of patient cardiac function

Regulation of heart development by the planar cell polarity pathway through the actomyosin complex and the mechanical forces

The heart is the first functional organ to form during vertebrate development and it is evolutionarily conserved across species. It is crucial for the proper delivering of essential nutrients and oxygen throughout the embryo's body. Its development is complex and requires fine-tuning processes at levels involving growth, differentiation, and morphogenesis. First, a linear heart tube is formed, followed by cardiac looping, chamber formation, and maturation. As for many organs, the heart arises from a simple epithelium with planar polarity properties. The genetic and molecular programs involved in heart formation have been studied for a long time. However, besides the genetic and cellular contributions to heart formation, little is known about the molecular and cellular components involved in generating tissue and tension forces required in heart morphogenesis. Embryonic heart tube remodeling requires coordination of actomyosin-dependent tissue forces fundamental to the emergence of cardiac chambers and looped heart. It has been established that cardiac chamber remodeling is coordinated through tissue-scale polarization of actomyosin. Here, using zebrafish as a model, I describe the role of actomyosin in generating and distributing the tension forces necessary across the ventricular myocardium during cardiac looping and chamber formation. I describe the spatiotemporal distribution of phosphorylated myosin during embryonic heart formation. A mathematical model was generated to demonstrate that the tissue-scale supracellular polarization of actomyosin within the myocardial epithelium is essential for heart formation. The mathematical model serves as a predictive tool of cardiac looping and chamber formation and supports its dependence on the proper actomyosin distribution. Examining the molecular mechanisms governing the actomyosin activity along the heart tube, I demonstrate that both Rho-associated Protein Kinase 2a (Rock2a) and cardiac-specific Myosin Light Chain Kinase 3 (Mylk3) regulate the actomyosin-based tissue forces through the phosphorylation state of the Myosin Regulatory Light Chain (MRLC). I show that the preferential basal activity of Mylk3 and the apical activity of Rock2a mediate the proper levels of phosphorylated myosin (pMyo) and its polarized distribution along the apicobasal axis within the myocardium. I propose that the antagonistic force-generating activities of Mylk3 and Rock2a facilitate mechano-molecular control of heart tube morphogenesis. Moreover, I show Mylk3 and Rock2a are under the genetic control of Planar Cell Polarity signaling, identifying Mylk3 as a novel tissue-specific effector, downstream of the Vangl2 branch of this signaling pathway. Altogether, these findings describe for the first time a mechano-molecular mechanism necessary for proper looping and chamber formation during heart development

Insight into the Gating Mechanism of Mechanosensitive Ion Channels using a simple structure: A step in the analysis of commotio cordis

Mechanosensation in cells is a well known phenomenon that is associated with cellular responses to force. Our knowledge of the trigger mechanism of this phenomenon is, however, limited. Earlier studies in this field have used atomic simulations, which although being accurate, are limited in their feasibility in multi-length scenarios like a mechanosensitive channel that undergoes micro-level changes in the composition of the protein to cause a macro-level change in the state of a biological structure such as the muscle. Finite Element Analysis has been used in various engineering fields to study the mechanical response of complex structures. The current study is a step in utilizing the phenomenal capabilities of Finite Element Analysis in developing and studying a 3D model (Membrane-Channel) of a mechanosensitive channel of large conductance (MscL). A simplified CAD structure of Mycobacterium tuberculosis (TbMscL) was developed in the first stage of this study. The authenticity of this model was tested by applying two types of loading conditions, namely (i) In-plane stretch and (ii) Out-of-plane bending. The results obtained from the first step of analysis are in accordance with previous experimental data, which elucidates the fact that tension within the membrane guides the gating mechanism of the channel and not the curvature of the membrane. The second stage of the analysis involved the use of the same model to study the disease commotio cordis. This was achieved by calculating the loading conditions during the onset of the condition in the human heart and then transferring those conditions to the Membrane-Channel model developed in the first stage. The result showed that although the channel did not fully open but there was a significant change in the channel‟s radius that might cause the flow of ions, thereby changing the state of the channel. It is anticipated that this model will help future research in areas that conventionally have been difficult to model