Francesco S. Pasqualini

A Novel Computational Model of the Human Ventricular Action Potential and Ca Transient

We have developed a detailed mathematical model for Ca handling and ionic currents in the human ventricular myocyte. Our aims were to: (1) simulate basic excitation-contraction coupling phenomena; (2) use realistic repolarizing K current densities; (3) reach steady-state. The model relies on the framework of the rabbit myocyte model previously developed by our group, with subsarcolemmal and junctional compartments where ion channels sense higher [Ca] vs. bulk cytosol. Ion channels and transporters have been modeled on the basis of the most recent experimental data from human ventricular myocytes. Rapidly and slowly inactivating components of I(to) have been formulated to differentiate between endocardial and epicardial myocytes. Transmural gradients of Ca handling proteins and Na pump were also simulated. The model has been validated against a wide set of experimental data including action potential duration (APD) adaptation and restitution, frequency-dependent increase in Ca transient peak and [Na](i). Interestingly, Na accumulation at fast heart rate is a major determinant of APD shortening, via outward shifts in Na pump and Na-Ca exchange currents. We investigated the effects of blocking K currents on APD and repolarization reserve: I(Ks) block does not affect the former and slightly reduces the latter; I(K1) blockade modestly increases APD and more strongly reduces repolarization reserve; I(Kr) blockers significantly prolong APD, an effect exacerbated as pacing frequency is decreased, in good agreement with experimental results in human myocytes. We conclude that this model provides a useful framework to explore excitation-contraction coupling mechanisms and repolarization abnormalities at the single myocyte level.

Instrumented cardiac microphysiological devices via multimaterial three-dimensional printing

Biomedical research has relied on animal studies and conventional cell cultures for decades. Recently, microphysiological systems (MPS), also known as organs-on-chips, that recapitulate the structure and function of native tissues in vitro, have emerged as a promising alternative1. However, current MPS typically lack integrated sensors and their fabrication requires multi-step lithographic processes2. Here, we introduce a facile route for fabricating a new class of instrumented cardiac microphysiological devices via multi-material 3D printing. Specifically, we designed six functional inks, based on piezo-resistive, high conductance, and biocompatible soft materials that enable integration of soft strain gauge sensors within micro-architectures that guide the self-assembly of physio-mimetic laminar cardiac tissues. We validated that these embedded sensors provide non-invasive, electronic readout of tissue contractile stresses, inside cell incubator environments. We further applied these devices to study drug responses, as well as the contractile development of human stem cell derived laminar cardiac tissues over four weeks.

Phototactic guidance of a tissue-engineered soft-robotic ray

Swim into the light A bio-inspired swimming robot that mimics a ray fish can be guided by light. Park et al. built a 1/10th-scale version of a ray fish with a microfabricated gold skeleton and a rubber body powered by rat heart muscle cells. The cardiomyocytes were genetically engineered to respond to light cues, so that the undulatory movements propelling the robot through water would follow a light source. Science, this issue p. 158 A biohybrid swimming robot powered by cardiomyocytes is optogenetically engineered to respond to light cues. Inspired by the relatively simple morphological blueprint provided by batoid fish such as stingrays and skates, we created a biohybrid system that enables an artificial animal—a tissue-engineered ray—to swim and phototactically follow a light cue. By patterning dissociated rat cardiomyocytes on an elastomeric body enclosing a microfabricated gold skeleton, we replicated fish morphology at 110 scale and captured basic fin deflection patterns of batoid fish. Optogenetics allows for phototactic guidance, steering, and turning maneuvers. Optical stimulation induced sequential muscle activation via serpentine-patterned muscle circuits, leading to coordinated undulatory swimming. The speed and direction of the ray was controlled by modulating light frequency and by independently eliciting right and left fins, allowing the biohybrid machine to maneuver through an obstacle course.

Quality Metrics for Stem Cell-Derived Cardiac Myocytes

Summary Advances in stem cell manufacturing methods have made it possible to produce stem cell-derived cardiac myocytes at industrial scales for in vitro muscle physiology research purposes. Although FDA-mandated quality assurance metrics address safety issues in the manufacture of stem cell-based products, no standardized guidelines currently exist for the evaluation of stem cell-derived myocyte functionality. As a result, it is unclear whether the various stem cell-derived myocyte cell lines on the market perform similarly, or whether any of them accurately recapitulate the characteristics of native cardiac myocytes. We propose a multiparametric quality assessment rubric in which genetic, structural, electrophysiological, and contractile measurements are coupled with comparison against values for these measurements that are representative of the ventricular myocyte phenotype. We demonstrated this procedure using commercially available, mass-produced murine embryonic stem cell- and induced pluripotent stem cell-derived myocytes compared with a neonatal mouse ventricular myocyte target phenotype in coupled in vitro assays.

Origin of Cardiomyocytes in the Adult Heart

This review article discusses the mechanisms of cardiomyogenesis in the adult heart. They include the re-entry of cardiomyocytes into the cell cycle; dedifferentiation of pre-existing cardiomyocytes, which assume an immature replicating cell phenotype; transdifferentiation of hematopoietic stem cells into cardiomyocytes; and cardiomyocytes derived from activation and lineage specification of resident cardiac stem cells. The recognition of the origin of cardiomyocytes is of critical importance for the development of strategies capable of enhancing the growth response of the myocardium; in fact, cell therapy for the decompensated heart has to be based on the acquisition of this fundamental biological knowledge.

Structural Phenotyping of Stem Cell-Derived Cardiomyocytes

Summary Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes.

Theoretical investigation of action potential duration dependence on extracellular Ca2+ in human cardiomyocytes

Reduction in [Ca2+]o prolongs the AP in ventricular cardiomyocytes and the QTc interval in patients. Although this phenomenon is relevant to arrhythmogenesis in the clinical setting, its mechanisms are counterintuitive and incompletely understood. To evaluate in silico the mechanisms of APD modulation by [Ca2+]o in human cardiomyocytes. We implemented the Ten Tusscher-Noble-Noble-Panfilov model of the human ventricular myocyte and modified the formulations of the rapidly and slowly activating delayed rectifier K+ currents (IKr and IKs) and L-type Ca2+ current (ICaL) to incorporate their known sensitivity to intra- or extracellular Ca2+. Simulations were run with the original and modified models at variable [Ca2+]o in the clinically relevant 1 to 3 mM range. The original model responds with APD shortening to decrease in [Ca2+]o, i.e. opposite to the experimental observations. Incorporation of Ca2+ dependency of K+ currents cannot reproduce the inverse relation between APD and [Ca2+]o. Only when ICaL inactivation process was modified, by enhancing its dependency on Ca2+, simulations predict APD prolongation at lower [Ca2+]o. Although Ca2+-dependent ICaL inactivation is the primary mechanism, secondary changes in electrogenic Ca2+ transport (by Na+/Ca2+ exchanger and plasmalemmal Ca2+-ATPase) contribute to the reversal of APD dependency on [Ca2+]o. This theoretical investigation points to Ca2+-dependent inactivation of ICaL as a mechanism primarily responsible for the dependency of APD on [Ca2+]o. The modifications implemented here make the model more suitable to analyze repolarization mechanisms when Ca2+ levels are altered.

Matrix elasticity regulates the optimal cardiac myocyte shape for contractility

Concentric hypertrophy is characterized by ventricular wall thickening, fibrosis, and decreased myocyte length-to-width aspect ratio. Ventricular thickening is considered compensatory because it reduces wall stress, but the functional consequences of cell shape remodeling in this pathological setting are unknown. We hypothesized that decreases in myocyte aspect ratio allow myocytes to maximize contractility when the extracellular matrix becomes stiffer due to conditions such as fibrosis. To test this, we engineered neonatal rat ventricular myocytes into rectangles mimicking the 2-D profiles of healthy and hypertrophied myocytes on hydrogels with moderate (13 kPa) and high (90 kPa) elastic moduli. Actin alignment was unaffected by matrix elasticity, but sarcomere content was typically higher on stiff gels. Microtubule polymerization was higher on stiff gels, implying increased intracellular elastic modulus. On moderate gels, myocytes with moderate aspect ratios (∼7:1) generated the most peak systolic work compared with other cell shapes. However, on stiffer gels, low aspect ratios (∼2:1) generated the most peak systolic work. To compare the relative contributions of intracellular vs. extracellular elasticity to contractility, we developed an analytical model and used our experimental data to fit unknown parameters. Our model predicted that matrix elasticity dominates over intracellular elasticity, suggesting that the extracellular matrix may potentially be a more effective therapeutic target than microtubules. Our data and model suggest that myocytes with lower aspect ratios have a functional advantage when the elasticity of the extracellular matrix decreases due to conditions such as fibrosis, highlighting the role of the extracellular matrix in cardiac disease.

Late Na + current and protracted electrical recovery are critical determinants of the aging myopathy

The aging myopathy manifests itself with diastolic dysfunction and preserved ejection fraction. We raised the possibility that, in a mouse model of physiological aging, defects in electromechanical properties of cardiomyocytes are important determinants of the diastolic characteristics of the myocardium, independently from changes in structural composition of the muscle and collagen framework. Here we show that an increase in the late Na+ current (INaL) in aging cardiomyocytes prolongs the action potential (AP) and influences temporal kinetics of Ca2+ cycling and contractility. These alterations increase force development and passive tension. Inhibition of INaL shortens the AP and corrects dynamics of Ca2+ transient, cell contraction and relaxation. Similarly, repolarization and diastolic tension of the senescent myocardium are partly restored. Thus, INaL offers inotropic support, but negatively interferes with cellular and ventricular compliance, providing a new perspective of the biology of myocardial aging and the aetiology of the defective cardiac performance in the elderly.

The contractile strength of vascular smooth muscle myocytes is shape dependent

Vascular smooth muscle cells in muscular arteries are more elongated than those in elastic arteries. Previously, we reported changes in the contractility of engineered vascular smooth muscle tissue that appeared to be correlated with the shape of the constituent cells, supporting the commonly held belief that elongated muscle geometry may allow for the better contractile tone modulation required in response to changes in blood flow and pressure. To test this hypothesis more rigorously, we developed an in vitro model by engineering human vascular smooth muscle cells to take on the same shapes as those seen in elastic and muscular arteries and measured their contraction during stimulation with endothelin-1. We found that in the engineered cells, actin alignment and nuclear eccentricity increased as the shape of the cell elongated. Smooth muscle cells with elongated shapes exhibited lower contractile strength but greater percentage increase in contraction after endothelin-1 stimulation. We analysed the relationship between smooth muscle contractility and subcellular architecture and found that changes in contractility were correlated with actin alignment and nuclear shape. These results suggest that elongated smooth muscle cells facilitate muscular artery tone modulation by increasing its dynamic contractile range.