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We present the NetSci program–an open-source scientific software package designed for estimating mutual information (MI) between data sets using GPU acceleration and a k-nearest-neighbor algorithm. This approach significantly enhances calculation speed, achieving improvements of several orders of magnitude over traditional CPU-based methods, with data set size limits dictated only by available hardware. To validate NetSci, we accurately compute MI for an analytically verifiable two-dimensional Gaussian distribution and replicate the generalized correlation (GC) analysis previously conducted on the B1 domain of protein G. We also apply NetSci to molecular dynamics simulations of the Sarcoendoplasmic Reticulum Calcium-ATPase (SERCA) pump, exploring the allosteric mechanisms and pathways influenced by ATP and 2′-deoxy-ATP (dATP) binding. Our analysis reveals distinct allosteric effects induced by ATP compared to dATP, with predicted information pathways from the bound nucleotide to the calcium-binding domain differing based on the nucleotide involved. NetSci proves to be a valuable tool for estimating MI and GC in various data sets and is particularly effective for analyzing intraprotein communication and information transfer.

Electrical impulse generation and its conduction within cells or cellular networks are the cornerstone of electrophysiology. However, the advancement of the field is limited by sensing accuracy and the scalability of current recording technologies. Here we describe a scalable platform that enables accurate recording of transmembrane potentials in electrogenic cells. The platform employs a three-dimensional high-performance field-effect transistor array for minimally invasive cellular interfacing that produces faithful recordings, as validated by the gold standard patch clamp. Leveraging the high spatial and temporal resolutions of the field-effect transistors, we measured the intracellular signal conduction velocity of a cardiomyocyte to be 0.182 m s−1, which is about five times the intercellular velocity. We also demonstrate intracellular recordings in cardiac muscle tissue constructs and reveal the signal conduction paths. This platform could provide new capabilities in probing the electrical behaviours of single cells and cellular networks, which carries broad implications for understanding cellular physiology, pathology and cell–cell interactions.

Supplemental Digital Content is available in the text.Heart failure with preserved ejection fraction (HFpEF), a major public health problem that is rising in prevalence, is associated with high morbidity and mortality and is considered to be the greatest unmet need in cardiovascular medicine today because of a general lack of effective treatments. To address this challenging syndrome, the National Heart, Lung, and Blood Institute convened a working group made up of experts in HFpEF and novel research methodologies to discuss research gaps and to prioritize research directions over the next decade. Here, we summarize the discussion of the working group, followed by key recommendations for future research priorities. There was uniform recognition that HFpEF is a highly integrated, multiorgan, systemic disorder requiring a multipronged investigative approach in both humans and animal models to improve understanding of mechanisms and treatment of HFpEF. It was recognized that advances in the understanding of basic mechanisms and the roles of inflammation, macrovascular and microvascular dysfunction, fibrosis, and tissue remodeling are needed and ideally would be obtained from (1) improved animal models, including large animal models, which incorporate the effects of aging and associated comorbid conditions; (2) repositories of deeply phenotyped physiological data and human tissue, made accessible to researchers to enhance collaboration and research advances; and (3) novel research methods that take advantage of computational advances and multiscale modeling for the analysis of complex, high-density data across multiple domains. The working group emphasized the need for interactions among basic, translational, clinical, and epidemiological scientists and across organ systems and cell types, leveraging different areas or research focus, and between research centers. A network of collaborative centers to accelerate basic, translational, and clinical research of pathobiological mechanisms and treatment strategies in HFpEF was discussed as an example of a strategy to advance research progress. This resource would facilitate comprehensive, deep phenotyping of a multicenter HFpEF patient cohort with standardized protocols and a robust biorepository. The research priorities outlined in this document are meant to stimulate scientific advances in HFpEF by providing a road map for future collaborative investigations among a diverse group of scientists across multiple domains.

Heterogeneous mechanical dyskinesis has been implicated in many arrhythmogenic phenotypes. Strain-dependent perturbations to cardiomyocyte electrophysiology may contribute to this arrhythmogenesis through processes referred to as mechanoelectric feedback. Although the role of stretch-activated ion currents has been investigated using computational models, experimental studies suggest that mechanical strain may also promote arrhythmia by facilitating calcium wave propagation. To investigate whether strain-dependent changes in calcium affinity to the myofilament may promote arrhythmogenic intracellular calcium waves, we modified a mathematical model of rabbit excitation-contraction coupling coupled to a model of myofilament activation and force development. In a one-dimensional compartmental analysis, we bidirectionally coupled 50 sarcomere models in series to model calcium diffusion and stress transfer between adjacent sarcomeres. These considerations enabled the model to capture 1) the effects of mechanical feedback on calcium homeostasis at the sarcomeric level and 2) the combined effects of mechanical and calcium heterogeneities at the cellular level. The results suggest that in conditions of calcium overload, the vulnerable window of stretch-release to trigger suprathreshold delayed afterdepolarizations can be affected by heterogeneity in sarcomere length. Furthermore, stretch and sarcomere heterogeneity may modulate the susceptibility threshold for delayed afterdepolarizations and the aftercontraction wave propagation velocity.

We use Brownian-Langevin dynamics principles to derive a coarse-graining multiscale myofilament model that can describe the thin-filament activation process during contraction. The model links atomistic molecular simulations of protein-protein interactions in the thin-filament regulatory unit to sarcomere-level activation dynamics. We first calculate the molecular interaction energy between tropomyosin and actin surface using Brownian dynamics simulations. This energy profile is then generalized to account for the observed tropomyosin transitions between its regulatory stable states. The generalized energy landscape then served as a basis for developing a filament-scale model using Langevin dynamics. This integrated analysis, spanning molecular to thin-filament scales, is capable of tracking the events of the tropomyosin conformational changes as it moves over the actin surface. The tropomyosin coil with flexible overlap regions between adjacent tropomyosins is represented in the model as a system of coupled stochastic ordinary differential equations. The proposed multiscale approach provides a more detailed molecular connection between tropomyosin dynamics, the trompomyosin-actin interaction-energy landscape, and the generated force by the sarcomere.

Myocardial hypertrophy is the result of sustained perturbations to the mechanical and/or neurohormonal homeostasis of cardiac cells and is driven by integrated, multiscale biophysical and biochemical processes that are currently not well defined. In this brief review, we highlight recent computational and experimental models of cardiac hypertrophy that span mechanisms from the molecular level to the tissue level. Specifically, we focus on the following: (i) molecular-level models of the structural dynamics of sarcomere proteins in hypertrophic hearts, (ii) cellular-level models of excitation–contraction coupling and mechanosensitive signaling in disease-state myocytes, and (iii) organ-level models of myocardial growth kinematics and predictors thereof. Finally, we discuss how spanning these scales and combining multiple experimental/computational models will provide new information about the processes governing hypertrophy and potential methods to prevent or reverse them.

The intact heart undergoes complex and multiscale remodelling processes in response to altered mechanical cues. Remodelling of the myocardium is regulated by a combination of myocyte and non-myocyte responses to mechanosensitive pathways, which can alter gene expression and therefore function in these cells. Cellular mechanotransduction and its downstream effects on gene expression are initially compensatory mechanisms during adaptations to the altered mechanical environment, but under prolonged and abnormal loading conditions, they can become maladaptive, leading to impaired function and cardiac pathologies. In this Review, we summarize mechanoregulated pathways in cardiac myocytes and fibroblasts that lead to altered gene expression and cell remodelling under physiological and pathophysiological conditions. Developments in systems modelling of the networks that regulate gene expression in response to mechanical stimuli should improve integrative understanding of their roles in vivo and help to discover new combinations of drugs and device therapies targeting mechanosignalling in heart disease.

Computational models and experimental optical mapping of cardiac electrophysiology serve as powerful tools to investigate the underlying mechanisms of arrhythmias. Modeling can also aid the interpretation of optical mapping signals, which may have different characteristics with respect to the underlying electrophysiological signals they represent. However, despite the prevalence of atrial arrhythmias such as atrial fibrillation, models of optical electrical mapping incorporating realistic structure of the atria are lacking. Therefore, we developed image-based models of atrial tissue using structural information extracted from optical coherence tomography (OCT), which can provide volumetric tissue characteristics in high resolution. OCT volumetric data of four swine atrial tissue samples were used to develop models incorporating tissue geometry, tissue-specific myofiber orientation, and ablation lesion regions. We demonstrated the use of these models through electrophysiology and photon scattering simulations. Changes in transmural electrical conduction were observed with the inclusion of OCT-derived, depth-resolved fiber orientation. Additionally, the amplitude of optical mapping signals were not found to correspond with lesion transmurality because of lesion geometry and electrical propagation occurring beyond excitation light penetration. This work established a framework for the development of tissue-specific models of atrial tissue derived from OCT imaging data, which can be useful in future investigations of electrophysiology and optical mapping signals with respect to realistic atrial tissue structure.

Cardiac fibroblasts are essential for beneficial myocardial healing but also cause detrimental adverse remodeling following myocardial infarction. The mechanical properties of the infarcted myocardium and border regions display temporal and spatial characteristics that regulate different aspects of the profibrotic cardiac fibroblast phenotypes.

Cardiac shape modeling is a useful computational tool that has provided quantitative insights into the mechanisms underlying dysfunction in heart disease. The manual input and time required to make cardiac shape models, however, limits their clinical utility. Here we present an end-to-end pipeline that uses deep learning for automated view classification, slice selection, phase selection, anatomical landmark localization, and myocardial image segmentation for the automated generation of three-dimensional, biventricular shape models. With this approach, we aim to make cardiac shape modeling a more robust and broadly applicable tool that has processing times consistent with clinical workflows.

Introduction:Troponin inhibitor3 (TNNI3) is a thin-filament protein that regulates contraction of thick filaments. The role of the switch domain of TNNI3(aa147-163), which interacts with the calcium-binding pocket of troponin C, is poorly defined. Pathogenic mutations to the switch domain cause restrictive cardiomyopathy in humans, but no therapies exist that address the underlying problem of this mutation at the sarcomeric level. Further, few models of genetic restrictive cardiomyopathy exist to aid in development of new therapies.Hypothesis:Substitution of alanine with valine at position 157 (A157V) in the switch domain of TNNI3causes restrictive cardiomyopathy by blunting response to adrenergic stimulus.Methods:A known pathogenic mutation to the TNNI3switch domain (A157V) was identified in a family of patients with cardiomyopathy and restrictive features. A mutant knock-in mouse homozygous for this mutation (A157V) was generated using CRISPR-Cas9 and used to elucidate the function of the switch domain.Results:Compared to wild type controls (WT), mutant A157V mice demonstrate significant restrictive features on invasive hemodynamics that worsen with age but do not show evidence of systolic dysfunction or hypertrophy on echocardiography. Heart size and myocyte cross-sectional area were significantly smaller in mutant A157V mice compared to WT controls. Molecular dynamics simulations revealed reduced TNNI3activation in response to PKA-mediated phosphorylation at serine23/24. Isolated myocytes from A157V mice demonstrated impaired relaxation, lower peak systolic calcium and delayed reuptake of calcium into the sarcoplasmic reticulum compared to WT controls.Conclusions:The A157V mutation to the switch domain of TNNI3, a critical regulatory domain that interacts with the calcium binding pocket of troponin C, causes diastolic dysfunction by impairing responsiveness to PKA-mediated phosphorylation of S23/24. This mouse model recapitulates the key restrictive features of human disease and could be used as a platform to study future targeted therapeutics for thin filament cardiomyopathy.

The molecular pharmacology of the G protein–coupled receptors for sphingosine 1-phosphate (S1P) provides important insight into established and new therapeutic targets. A new, potent bitopic S1P3antagonist, SPM-354, with in vivo activity, has been used, together with S1P3-knockin and S1P3-knockout mice to define the spatial and functional properties of S1P3in regulating cardiac conduction. We show that S1P3is a key direct regulator of cardiac rhythm both in vivo and in isolated perfused hearts. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol in vivo and S1P in isolated hearts induced a spectrum of cardiac effects, ranging from sinus bradycardia to complete heart block, as measured by a surface electrocardiogram in anesthetized mice and in volume-conducted Langendorff preparations. The agonist effects on complete heart block are absent in S1P3-knockout mice and are reversed in wild-type mice with SPM-354, as characterized and described here. Homologous knockin of S1P3-mCherry is fully functional pharmacologically and is strongly expressed by immunohistochemistry confocal microscopy in Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4)-positive atrioventricular node and His-Purkinje fibers, with relative less expression in the HCN4-positive sinoatrial node. In Langendorff studies, at constant pressure, SPM-354 restored sinus rhythm in S1P-induced complete heart block and fully reversed S1P-mediated bradycardia. S1P3distribution and function in the mouse ventricular cardiac conduction system suggest a direct mechanism for heart block risk that should be further studied in humans. A richer understanding of receptor and ligand usage in the pacemaker cells of the cardiac system is likely to be useful in understanding ventricular conduction in health, disease, and pharmacology.

Supplemental Digital Content is available in the text.

During β-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease KCaand pCa50, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow), which speeds up the overall relaxation. However, as the N-terminus of cTnI (residues 1–40) has not been resolved in the whole cardiac troponin (cTn) structure, little is known about the molecular-level behavior within the whole cTn complex upon phosphorylation of the S23/S24 residues of cTnI that results in these changes in function. In this study, we built up the cTn complex structure (including residues cTnC 1–161, cTnI 1–172, and cTnT 236–285) with the N-terminus of cTnI. We performed molecular-dynamics (MD) simulations to elucidate the structural basis of PKA phosphorylation-induced changes in cTn structure and Ca2+binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca2+binding site II. However, the overall fluctuation of cTn was increased and the C-I interaction was altered relative to the wild-type model. The most significant changes involved interactions with the N-terminus of cTnI. Interestingly, the phosphomimic mutations led to the formation of intrasubunit interactions between the N-terminus and the inhibitory peptide of cTnI. This may result in altered interactions with cTnC and could explain the increased rate and decreased duration of slow-phase relaxation seen in myofibrils.

Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca2+levels vary. Troponin C (cTnC) interaction with cTnI (C-I interaction) is a critical step in contractile activation that can be modulated by cTnI phosphorylation. We tested the hypothesis that altering C-I interactions by PKA, or by cTnI phosphomimetic mutations (S23D/S24D-cTnI), directly affects thin filament activation and myofilament relaxation kinetics. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild-type cTnI, or S23D/S24D-cTnI recombinant cTn. Contractile mechanics were monitored at maximum and submaximal Ca2+concentrations. PKA treatment of wild-type cTn or exchange of cTn containing S23D/S24D-cTnI resulted in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow). These effects were greater for submaximal Ca2+activated contractions. PKA treatment also reduced the rate of contractile activation (kACT) at maximal, but not submaximal Ca2+, and reduced the Ca2+sensitivity of contraction. Using a fluorescent probe coupled to cTnC (C35S-IANBD), the Ca2+-cTn binding affinity and C-I interaction were monitored. Ca2+binding to cTn (pCa50) was significantly decreased when cTnI was phosphorylated by PKA (ΔpCa50 = 0.31). PKA phosphorylation of cTnI also weakened C-I interaction in the presence of Ca2+. These data suggest that weakened C-I interaction, via PKA phosphorylation of cTnI, may slow thin filament activation and result in increased myofilament relaxation kinetics, the latter of which could enhance early phase diastolic relaxation during β-adrenergic stimulation.

Contractile function of cardiac cells is driven by the sliding displacement of myofilaments powered by the cycling myosin crossbridges. Critical to this process is the availability of ATP, which myosin hydrolyzes during the cross-bridge cycle. The diffusion of adenine nucleotides through the myofilament lattice has been shown to be anisotropic, with slower radial diffusion perpendicular to the filament axis relative to parallel, and is attributed to the periodic hexagonal arrangement of the thin (actin) and thick (myosin) filaments. We investigated whether atomistic-resolution details of myofilament proteins can refine coarse-grain estimates of diffusional anisotropy for adenine nucleotides in the cardiac myofibril, using homogenization theory and atomistic thin filament models from the Protein Data Bank. Our results demonstrate considerable anisotropy in ATP and ADP diffusion constants that is consistent with experimental measurements and dependent on lattice spacing and myofilament overlap. A reaction-diffusion model of the half-sarcomere further suggests that diffusional anisotropy may lead to modest adenine nucleotide gradients in the myoplasm under physiological conditions.

Introduction:Calcium-calmodulin-dependent protein kinase II (CaMKII) has emerged as a central mediator of cardiac stress responses which may serve several critical roles in the regulation of cardiac rhythm, cardiac contractility and growth. Sustained and excessive activation of CaMKII during cardiac disease has, however, been linked to arrhythmias, and maladaptive cardiac remodeling, eventually leading to heart failure (HF) and sudden cardiac death.Areas covered:In the current review, the authors describe the unique structural and biochemical properties of CaMKII and focus on its physiological effects in cardiomyocytes. Furthermore, they provide evidence for a role of CaMKII in cardiac pathologies, including arrhythmogenesis, myocardial ischemia and HF development. The authors conclude by discussing the potential for CaMKII as a target for inhibition in heart disease.Expert opinion:CaMKII provides a promising nodal point for intervention that may allow simultaneous prevention of HF progression and development of arrhythmias. For future studies and drug development there is a strong rationale for the development of more specific CaMKII inhibitors. In addition, an improved understanding of the differential roles of CaMKII subtypes is required.

Vinculin (Vcl) plays a key structural role in ventricular myocytes that, when disrupted, can lead to contractile dysfunction and dilated cardiomyopathy. To investigate the role of Vcl in myocyte and myocardial function, cardiomyocyte-specific Vcl knockout mice (cVclKO) and littermate control wild-type mice were studied with transmission electron microscopy (TEM) and in vivo magnetic resonance imaging (MRI) tagging before the onset of global ventricular dysfunction. MRI revealed significantly decreased systolic strains transverse to the myofiber axis in vivo, but no changes along the muscle fibers or in fiber tension in papillary muscles from heterozygous global Vcl null mice. Myofilament lattice spacing from TEM was significantly greater in cVclKO versus wild-type hearts fixed in the unloaded state. AFM in Vcl heterozygous null mouse myocytes showed a significant decrease in membrane cortical stiffness. A multiscale computational model of ventricular mechanics incorporating cross-bridge geometry and lattice mechanics showed that increased transverse systolic stiffness due to increased lattice spacing may explain the systolic wall strains associated with Vcl deficiency, before the onset of ventricular dysfunction. Loss of cardiac myocyte Vcl may decrease systolic transverse strains in vivo by decreasing membrane cortical tension, which decreases transverse compression of the lattice thereby increasing interfilament spacing and stress transverse to the myofibers.

Familial hypertrophic cardiomyopathy (FHC) is an inherited disorder affecting roughly one in 500 people. Its hallmark is abnormal thickening of the ventricular wall, leading to serious complications that include heart failure and sudden cardiac death. Treatment is complicated by variation in the severity, symptoms and risks for sudden death within the patient population. Nearly all of the genetic lesions associated with FHC occur in genes encoding sarcomeric proteins, indicating that defects in cardiac muscle contraction underlie the condition. Detailed biophysical data are increasingly available for computational analyses that could be used to predict heart phenotypes based on genotype. These models must integrate the dynamic processes occurring in cardiac cells with properties of myocardial tissue, heart geometry and haemodynamic load in order to predict strain and stress in the ventricular walls and overall pump function. Recent advances have increased the biophysical detail in these models at the myofilament level, which will allow properties of FHC-linked mutant proteins to be accurately represented in simulations of whole heart function. The short-term impact of these models will be detailed descriptions of contractile dysfunction and altered myocardial strain patterns at the earliest stages of the disease—predictions that could be validated in genetically modified animals. Long term, these multi-scale models have the potential to improve clinical management of FHC through genotype-based risk stratification and personalized therapy.

Heart failure (HF) in combination with mechanical dyssynchrony is associated with a high mortality rate. To quantify contractile dysfunction in patients with HF, investigators have proposed several indices of mechanical dyssynchrony, including percentile range of time to peak shortening (WTpeak), circumferential uniformity ratio estimate (CURE), and internal stretch fraction (ISF). The goal of this study was to compare the sensitivity of these indices to 4 major abnormalities responsible for cardiac dysfunction in dyssynchronous HF: dilation, negative inotropy, negative lusitropy, and dyssynchronous activation.

We developed a Markov model of cardiac thin filament activation that accounts for interactions among nearest-neighbor regulatory units (RUs) in a spatially explicit manner. Interactions were assumed to arise from structural coupling of adjacent tropomyosins (Tms), such that Tm shifting within each RU was influenced by the Tm status of its neighbors. Simulations using the model demonstrate that this coupling is sufficient to produce observed cooperativity in both steady-state and dynamic force-Ca2+relationships. The model was further validated by comparison with reported responses under various conditions including inhibition of myosin binding and the addition of strong-binding, non-force-producing myosin fragments. The model also reproduced the effects of 2.5 mM added Pion Ca2+-activated force and the rate of force redevelopment measured in skinned rat myocardial preparations. Model analysis suggests that Tm-Tm coupling potentiates the activating effects of strongly-bound cross-bridges and contributes to force-Ca2+dynamics of intact cardiac muscle. The model further predicts that activation at low Ca2+concentrations is cooperatively inhibited by nearest neighbors, requiring Ca2+binding to >25% of RUs to produce appreciable levels of force. Without excluding other putative cooperative mechanisms, these findings suggest that structural coupling of adjacent Tm molecules contributes to several properties of cardiac myofilament activation.

Effective therapy of complex diseases requires control of highly nonlinear complex networks that remain incompletely characterized. In particular, drug intervention can be seen as control of cellular network activity. Identification of control parameters presents an extreme challenge due to the combinatorial explosion of control possibilities in combination therapy and to the incomplete knowledge of the systems biology of cells. In this review paper, we describe the main current and proposed approaches to the design of combinatorial therapies, including the heuristic methods used now by clinicians and alternative approaches suggested recently by several authors. New approaches for designing combinations arising from systems biology are described. We discuss in special detail the design of algorithms that identify optimal control parameters in cellular networks based on a quantitative characterization of control landscapes, maximizing utilization of incomplete knowledge of the state and structure of intracellular networks. The use of new technology for high‐throughput measurements is key to these new approaches to combination therapy and essential for the characterization of control landscapes and implementation of the algorithms. Combinatorial optimization in medical therapy is also compared with the combinatorial optimization of engineering and materials science and similarities and differences are delineated. Copyright © 2009 John Wiley & Sons, Inc.

Cardiac remodeling is regulated at the cellular level by coordinated responses to biomechanical signals, such as actomyosin contractile forces, hemodynamic loading, and extracellular matrix (ECM) mechanical properties. In cardiac muscle cells (cardiomyocytes), the nucleus and the ECM are physically connected by a series of structural protein networks that span the cell membrane, the sarcomere, the cytoskeleton, and the nucleoskeleton. Consequently, prolonged abnormal forces (e.g. hypertension, fibrosis, protein variants) are ‘sensed’ by and propagated through these biological structures, ultimately inducing gene expression programs that mediate remodeling of cell and tissue structure. In this brief review, we discuss mechanisms by which biomechanical stimuli are transmitted and transduced into cellular responses that regulate cardiac phenotypes, focusing on cardiomyocyte remodeling in disease settings.

Cardiac cells mature in the first postnatal week, concurrent with altered extracellular mechanical properties. To investigate the effects of extracellular stiffness on cardiomyocyte maturation, we plated neonatal rat ventricular myocytes for 7 days on collagen-coated polyacrylamide gels with varying elastic moduli. Cells on 10kPa substrates developed aligned sarcomeres, whereas cells on stiffer substrates had unaligned sarcomeres and stress fibers, which are not observed in vivo. We found that cells generated greater mechanical force on gels with stiffness similar to the native myocardium, 10kPa, than on stiffer or softer substrates. Cardiomyocytes on 10kPa gels also had the largest calcium transients, sarcoplasmic calcium stores, and sarcoplasmic/endoplasmic reticular calcium ATPase2a expression, but no difference in contractile protein. We hypothesized that inhibition of stress fiber formation might allow myocyte maturation on stiffer substrates. Treatment of maturing cardiomyocytes with hydroxyfasudil, an inhibitor of RhoA kinase and stress fiber-formation, resulted in enhanced force generation on the stiffest gels. We conclude that extracellular stiffness near that of native myocardium significantly enhances neonatal rat ventricular myocytes maturation. Deviations from ideal stiffness result in lower expression of sarcoplasmic/endoplasmic reticular calcium ATPase, less stored calcium, smaller calcium transients, and lower force. On very stiff substrates, this adaptation seems to involve RhoA kinase.

We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo–like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.

Abstract Targeted deletion of actinin-associated LIM protein (ALP) in mice leads to right ventricular (RV) dysplasia and a mild RV cardiomyopathy. Although the phenotype has been thoroughly characterized, the mechanisms leading from the cytoskeletal defect to the disease are unclear. We hypothesized that ALP deficiency may be associated with (1) changes in regional systolic dysfunction and (2) regional dysregulation of hypertrophic growth, in accordance with the restricted expression of ALP in the outflow tract of the RV. We examined RV regional epicardial systolic strains with respect to end-diastole in ALP knockout (ALPKO) mice and wild-type controls using an open-chest preparation. Strain components were consistently lower in the ALPKO mice than wild-type controls (second principal strain E2: p = 0.05). RV pressure was slightly but not significantly lower in ALPKO mice as well. To assess regional growth, geometric remodeling was analyzed in ALPKO and wild-type mice after 4 weeks of chronic hypoxia (11% oxygen). The average amount of RV wall thickening in response to hypoxia was reduced to 11% in the ALPKO mice compared with 44% in the wild-type controls. In summary, the results are consistent with the view that disruption of ALP is associated with diminished RV contractile function as well as altered hypertrophic remodeling.

Cardiac myocytes and fibroblasts are essential elements of myocardial tissue structure and function. In vivo, myocytes constitute the majority of cardiac tissue volume, whereas fibroblasts dominate in numbers. In vitro, cardiac cell cultures are usually designed to exclude fibroblasts, which, because of their maintained proliferative potential, tend to overgrow the myocytes. Recent advances in microstructuring of cultures and cell growth on elastic membranes have greatly enhanced in vitro preservation of tissue properties and offer a novel platform technology for producing more in vivo-like models of myocardium. We used microfluidic techniques to grow two-dimensional structured cardiac tissue models, containing both myocytes and fibroblasts, and characterized cell morphology, distribution, and coupling using immunohistochemical techniques. In vitro findings were compared with in vivo ventricular cyto-architecture. Cardiac myocytes and fibroblasts, cultured on intersecting 30-μm-wide collagen tracks, acquire an in vivo-like phenotype. Their spatial arrangement closely resembles that observed in native tissue: Strands of highly aligned myocytes are surrounded by parallel threads of fibroblasts. In this in vitro system, fibroblasts form contacts with other fibroblasts and myocytes, which can support homogeneous and heterogeneous gap junctional coupling, as observed in vivo. We conclude that structured cocultures of cardiomyocytes and fibroblasts mimic in vivo ventricular tissue organization and provide a novel tool for in vitro research into cardiac electromechanical function.

Changes in cytosolic free Mg2+and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg2+, ATP, and ADP of KATPand L-type Ca2+channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the KATPcurrent and MgATP regulation of the L-type Ca2+current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on KATPcurrent, 2), simulates effects of ADP in modulating ATP sensitivity of KATPchannel, 3), predicts activation of Ca2+current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg2+or increased free Mg2+with normal ATP and ADP activate KATPcurrent, shorten action potential, and alter ionic currents and intracellular Ca2+signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

Objective:Magnesium regulates a large number of cellular processes. Small changes in intracellular free Mg2+([Mg2+]i) may have important effects on cardiac excitability and contractility. We investigated the effects of [Mg2+]ion cardiac excitation-contraction coupling.Methods:We used our ionic-metabolic model that incorporates equations for Ca2+and Mg2+buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na+-K+pump, sarcolemmal and sarcoplasmic Ca2+pumps).Results:Model results indicate that variations in cytosolic Mg2+level might sensitively affect diastolic and systolic Ca2+, sarcoplasmic Ca2+content, Ca2+influx through L-type channels, efficiency of the Na+/Ca2+exchanger and action potential shape. The analysis suggests that the most important reason for the observed effects is a modified normal function of sarcoplasmic Ca2+-ATPase pump by altered diastolic MgATP levels.Conclusion:The model is able to reproduce qualitatively a sequence of events that correspond well with experimental observations during cardiac excitation-contraction coupling in mammalian ventricular myocytes.

Mechanical interactions between desmin and Z-disks, costameres, and nuclei were measured during passive deformation of single muscle cells. Image processing and continuum kinematics were used to quantify the structural connectivity among these structures. Analysis of both wild-type and desmin-null fibers revealed that the costamere protein talin colocalized with the Z-disk protein α-actinin, even at very high strains and stresses. These data indicate that desmin is not essential for mechanical coupling of the costamere complex and the sarcomere lattice. Within the sarcomere lattice, significant differences in myofibrillar connectivity were revealed between passively deformed wild-type and desmin-null fibers. Connectivity in wild-type fibers was significantly greater compared to desmin-null fibers, demonstrating a significant functional connection between myofibrils that requires desmin. Passive mechanical analysis revealed that desmin may be partially responsible for regulating fiber volume, and consequently, fiber mechanical properties. Kinematic analysis of α-actinin strain fields revealed that knockout fibers transmitted less shear strain compared to wild-type fibers and experienced a slight increase in fiber volume. Finally, linkage of desmin intermediate filaments to muscle nuclei was strongly suggested based on extensive loss of nuclei positioning in the absence of desmin during passive fiber loading.

In the present study we have developed a multi-cue bioreactor (MCB) that is capable of delivering a range of stimuli to assist the development of a tissue-engineered construct. The MCB provides an accurate and utilizable computer-controlled pulsatile pump and strain induction mechanism and it has the capability of applying physiological conditions to samples. The device described here emulates the pressure and straining environment found at the aortic root. This function, along with an integral perfusion and sterile containment system, allows for long-term culture and whole-tissue testing capability. Aortic and pulmonary arteries were obtained from freshly isolated porcine hearts and subjected to various loading regimens (Δpressure/flow/force). Through analyzing data acquired by the MCB transducer array it was possible to differentiate the dynamic mechanical properties of the tissue types tested. In addition, the MCB illustrates a novel concept in cardiovascular tissue engineering: being able to support long-term tissue culture of cell-seeded substrates while they are under the influence of mechanical cues. After 7 days of pulsation in the MCB cell alignment was observed. The MCB represents a versatile model that will enable the development of tissue engineering not only for cardiovascular tissue, but for all tubular tissues such as esophageal, tracheal, and bronchial systems.

Because cell shape and alignment, cell–matrix adhesion, and cell–cell contact can all affect growth, and because mechanical strains in vivo are multiaxial and anisotropic, we developed an in vitro system for engineering aligned, rod-shaped, neonatal cardiac myocyte cultures. Photolithographic and microfluidic techniques were used to micropattern extracellular matrices in parallel lines on deformable silicone elastomers. Confluent, elongated, aligned myocytes were produced by varying the micropattern line width and collagen density. An elliptical cell stretcher applied 2:1 anisotropic strain statically to the elastic substrate, with the axis of greatest stretch (10%) either parallel or transverse to the myofibrils. After 24 h, the principal strain parallel to myocytes did not significantly alter myofibril accumulation or expression of atrial natriuretic factor (ANF), connexin-43 (Cx-43), or N-cadherin (by indirect immunofluorescent antibody labeling and immunoblotting) compared with unstretched controls. In contrast, 10% transverse principal strain resulted in continuous staining of actin filaments (rhodamine phalloidin); increased immunofluorescent labeling of ANF, Cx-43, and N-cadherin; and upregulation of protein signal intensity by western blotting. By using microfabrication and microfluidics to control cell shape and alignment on an elastic substrate, we found greater effects for transverse than for longitudinal stretch in regulating sarcomere organization, hypertrophy, and cell-to-cell junctions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 578–587, 2003.

The relations between stress, stimulation rate and sarcomere length (SL) were investigated in 24 cardiac trabeculae isolated from right ventricles of mice (CF‐1 males, 25‐30 g) and superfused with Hepes solution ([Ca2+]o= 1 mm, pH 7.4, 25 °C). Stress and SL were measured by a strain gauge transducer and laser diffraction technique, respectively. Stress versusstimulation frequency formed a biphasic relation (25 °C, [Ca2+]o= 2 mm) with a minimum at 0.7‐1 Hz (≈15 mN mm−2), a 150 % decrease from 0.1 to 1 Hz (descending limb) and a 75 % increase from 1 to 5 Hz (ascending limb). Ryanodine (0.1 μm) inhibited specifically the descending limb, while nifedipine (0.1 μm) affected specifically the ascending limb. This result suggests two separate sources of Ca2+for stress development: (1) net Ca2+influx during action potentials (AP); and (2) Ca2+entry into the cytosol from the extracellular space during diastolic intervals; Ca2+from both (1) and (2) is sequestered by the SR between beats. Raising the temperature to 37 °C lowered the stress‐frequency relation (SFR) by ≈0‐15 mN mm−2at each frequency. Because the amount of Ca2+carried by ICa,Lshowed a ≈3‐fold increase under the same conditions, we conclude that reduced Ca2+loading of the SR was probably responsible for this temperature effect. A simple model of Ca2+fluxes addressed the mechanisms underlying the SFR. Simulation of the effect of inorganic phosphates (Pi) on force production was incorporated into the model. The results suggested that O2diffusion limits force production at stimulation rates >3 Hz. The stress‐SL relations from slack length (≈1.75 μm) to 2.25 μm showed that the passive stress‐SL curve of mouse cardiac trabeculae is exponential with a steep increase at SL >2.1 μm. Active stress (at 1 Hz) increased with SL, following a curved relation with convexity toward the abscissa at [Ca2+]= 2 mm. At [Ca2+] from 4 to 12 mm, the stress‐SL curves superimposed and the relation became linear, which revealed a saturation step in the activation of force production. EC coupling in mouse cardiac muscle is similar to that observed previously in the rat, although important differences exist in the Ca2+dependence of force development. These results may suggest a lower capacity of the SR for buffering Ca2+, which makes the generation of force in mouse cardiac ventricle more dependent on Ca2+entering during action potentials, particularly at high heart rate.

Central to this analysis is the identification of six rotation invariant scalars α1–6 that succinctly define the strain in materials that have one family of parallel fibers arranged in laminae. These scalars were chosen so as to minimize covariance amongst the response terms in the hyperelastic limit, and they are termed strain attributes because it is necessary to distinguish them from strain invariants. The Cauchy stress t is expressed as the sum of six response terms, almost all of which are mutually orthogonal for finite strain (i.e. 14 of the 15 inner products vanish). For small deformations, the response terms are entirely orthogonal (i.e. all 15 inner products vanish). A response term is the product of a response function with its associated kinematic tensor. Each response function is a scalar partial derivative of the strain energy W with respect to a strain attribute. Applications for this theory presently include myocardium (heart muscle) which is often modeled as having muscle fibers arranged in sheets. Utility for experimental identification of strain energy functions is demonstrated by showing that common tests on incompressible materials can directly determine terms in W. Since the described set of strain attributes reduces the covariance amongst response terms, this approach may enhance the speed and precision of inverse finite element methods. [Copyright &y& Elsevier]

We coupled a continuum model of impulse propagation with a three-dimensional model of regional ventricular mechanics. Equations for action potential propagation, myofilament activation and active contraction were solved in an anatomically detailed finite element model of canine left and right ventricular geometry, muscle fiber architecture and Purkinje fiber network anatomy. Finite element equations for time-dependent excitation and recovery variables were assembled using a collocation method and solved using adaptive Runge–Kutta integration. Resting tissue mechanics were modeled as nonlinear, orthotropic and hyperelastic. A Windkessel model for arterial impedance was coupled to ventricular pressure and volume to compute hemodynamic boundary conditions during ejection. Ventricular volume constraints were imposed during the isovolumic phases. This model showed good agreement in the time course of regional systolic strains with experimental measurements during normal sinus rhythm and demonstrates the importance of the Purkinje fiber system in determining the mechanical activation sequence.

Introduction:Current indications for pulmonary valve replacement (PVR) in repaired tetralogy of Fallot (rTOF) rely on global ventricular measurements but are inconsistently applied, lead to mixed outcomes, and are widely debated.Hypothesis:We aimed to test the hypothesis that specific regional markers of biventricular shape can discriminate differences in ventricular remodeling between rTOF patients that were and were not designated for follow-up PVR better than standard clinical indices.Methods:In this cross-sectional retrospective study, biventricular shape models were fit to CMR images from 84 rTOF patients. A statistical atlas of end-diastolic shape was constructed using principal component analysis. Multivariate regression was used to quantify shape marker and clinical index associations with subsequent intervention status (PVR, n=48 vs. No-PVR, n=36) while accounting for confounders. Clustering analysis was then used to test the ability of statistically significant shape markers and clinical indices to discriminate PVR status as evaluated by Matthews correlation coefficient (MCC). Geometric strain analysis was also conducted to assess shape marker associations with systolic function.Results:PVR status correlated with shape markers associated with RV apical dilation and LV dilation (p<0.001), RV basal bulging and LV conicity (p<0.02), and pulmonary valve dilation (p<0.002). PVR status also correlated with RV EF (p<0.03) and non-significantly correlated with LV ESVi (p<0.06). Clustering analysis revealed that shape markers discriminated PVR status better than clinical indices (MCC=0.49 and MCC=0.28, respectively). These shape markers were also significantly associated with RV and LV radial strain.Conclusions:Biventricular shape markers discriminated PVR status better than standard and current use clinical indices for PVR. These specific, regional features of cardiac morphology can provide mechanistic insight into adaptive vs. maladaptive types of ventricular remodeling that provide the foundation for clinical decision-making but may be overlooked when relying solely on global ventricular measurements.

Quantitative assessment of myofiber disarray associated with diseases such as familial hypertrophic cardiomyopathy (FHC) can be performed by estimating local angular deviation of fiber orientation in histologic sections. The large number of measurements required to estimate angular deviation prohibits manual measurement. We describe methods for automated measurement of local orientation and angular deviation in tissue sections from transgenic mice with ventricular expression of ras, proposed as a model of FHC. Images of histologic tissue sections from normal and transgenic mice were analyzed using image processing techniques to estimate local orientation of myofibers. Results from the automated methods were compared with manual measurements. Automated methods estimated differing mean orientation in 7–20% of normal sections and 17–29% of transgenic tissue sections with differing dispersions in 23–30% of normal sections and 25% of transgenic tissue sections. Automated methods estimate 24.47 ± 13.03% of total ventricular mass affected by disarray that is comparable to a previous estimate of 21.7% in the same mouse model. Automated methods are a rapid and accurate alternative to manual measurement for estimation of mean orientation and angular deviation in myocardial tissue sections. Differences between manual and automated methods may be attributed to the substantially larger number of measurements made by automated methods. Automated methods are particularly appropriate for use in determining local variation in orientation such as focal myofiber disarray associated with FHC. The generality of these methods suggests they may have use in other biological fields such as quantifying cellular alignment. Anat Rec. 252:612–625, 1998. © 1998 Wiley-Liss, Inc.

Different patterns of extracellular matrix (ECM) remodeling in the heart are thought to be dependent on altered mechanical and chemical conditions and can contribute to cardiac dysfunction. Cardiac fibroblasts are the primary regulators of the ECM and may respond to mechanical factors in vitro. We hypothesized that different types of in vitrostrains, e.g. tensile or compressive, can stimulate different functional responses in cultured adult rat cardiac fibroblasts. In this study, we first showed that a single step in strain applied by a uniaxial stretch system stimulated collagen III and fibronectin mRNA levels and transforming growth factor- β1(TGF- β1) activity in the adult phenotype of rat cardiac fibroblasts. Two-dimensional deformations were measured by tracking fluorescent microspheres attached to the substrate and cultured cells. For 10% uniaxial strain, mean principal strains were 0.104±0.018 in the direction of stretch and −0.042±0.013 in the perpendicular direction, verifying that the fibroblasts were simultaneously subjected to tensile (positive) and compressive (negative) strains. Furthermore, these cells were also subjected to area change and to shear. In order to examine the distinct effects of different types of deformation on cardiac fibroblasts, an equibiaxial stretch system was used to apply either pure tensile or compressive area strains, in the absence of shear. Magnitudes of equibiaxial strain were selected to apply local cell area changes identical to those applied in the uniaxial system. Results showed that pure tensile and compressive area strains induced divergent responses in ECM mRNA levels. TGF-β1activity was dependent on the magnitude of applied area strain regardless of the mode of deformation. These findings demonstrate that adult cardiac fibroblasts may respond differently to varied types of mechanical loading, suggesting that ECM remodeling may be locally regulated by specific mechanical stimuli in the heart.

Quantitative assessment of myofiber disarray associated with diseases such as familial hypertrophic cardiomyopathy (FHC) can be performed by estimating local angular deviation of fiber orientation in histologic sections. The large number of measurements required to estimate angular deviation prohibits manual measurement. We describe methods for automated measurement of local orientation and angular deviation in tissue sections from transgenic mice with ventricular expression of ras,proposed as a model of FHC.

Cellular hypertrophy can alter the distribution of residual stress in the myocardium, hence can affect active and passive ventricular mechanics. It is hypothesized that an increase in stress-free cell cross-sectional area will tend to increase residual stresses. Therefore transmural distributions of myocyte cross-sectional areas and global ventricular dimensions in young rats 0–21 days following thoracic aortic banding with sham-operated and unoperated control groups were measured in tissue free of all external and residual stresses. Cell cross-sectional area increased in the stress-free state and was uniform across the wall except at 21 days when there was a transmural gradient, with cells at the endocardium 46% larger in diameter than those in the outer wall. Cell area increased from a mean of 156±30μm2at 0 days to a mean of 627±164μm2at 21 days, although during this time there were no statistical changes in the opening angles of stress-free tissue sections. Because the time course of opening angle did not follow the changes in cell thickening, the cellular growth measured in this study is probably not the only factor responsible for the distribution of residual stress.