A Heart Artificial: Building the Foundation for the Development and Maintenance of In Vitro Tissue Mimetic Cardiovascular Models

Given the prevalence of cardiovascular disorders and the distinct lack of significant repair mechanisms within cardiovascular systems, effective therapy for long-term treatment of cardiovascular degeneration remains a significant challenge. Further, the fundamental importance of such systems to all mammalian life begs the development of realistic component structures for in vitro assessment. Significant effort was expended to create in vitro models which mimicked a subset of structure and function of coordinate native components within cardiovascular systems. Towards this end, we developed a 3D-Artificial Heart Muscle (AHM) model utilizing fibrin gel and neonatal cardiac myocytes. We extracted functional metrics in order to probe the optimal protocol for generation of the tissue model. Building on the outcome of this experiment, we applied the optimal 3D-AHM model to a decellularized adult rat heart in order to re-append function to a complex acellular scaffold. The resultant bioartifical heart (BAH) model was assessed to identify the efficacy of 3D-AHM as a functional delivery mechanism and to lay a framework for heart model development. An alternative strategy for the generation of 3D heart muscle was explored through magnetic levitation of cardiovascular cells. Magnetic sensitivity was appended to cells through incubation with ferromagnetic nanoparticles. The cells were then levitated and cultured within a magnetic field to form 3D multicellular aggregates. (MCAs) We utilized a magnetized fibrin gel scaffold in order to apply non-contact, magnetic stretch conditioning to our AHM model through a novel bioreactor system. We were able to develop a highly functional 3D-AHM and extracted 4M cells as the optimal concentration for the generation of our artificial heart muscle. Application of a layer of 3D-AHM to an acellular rat heart proved the 3D-AHM an effective mechanism for delivery of a subset of function to a structure. Magnetic levitation generated hundreds of cell-dense, functional and phenotypically relevant heart muscle analogs. We have developed a completely novel system for the application of mechanical stretch conditioning to artificial heart muscle models and are working to implement more complex conditioning systems. The work presented herein surveys the generation of 3 unique cardiovascular model systems and a novel method for model conditioning.Biomedical Engineering, Department o

Cardiac Meets Skeletal: What's New in Microfluidic Models for Muscle Tissue Engineering

In the last few years microfluidics and microfabrication technique principles have been extensively exploited for biomedical applications. In this framework, organs-on-a-chip represent promising tools to reproduce key features of functional tissue units within microscale culture chambers. These systems offer the possibility to investigate the effects of biochemical, mechanical, and electrical stimulations, which are usually applied to enhance the functionality of the engineered tissues. Since the functionality of muscle tissues relies on the 3D organization and on the perfect coupling between electrochemical stimulation and mechanical contraction, great efforts have been devoted to generate biomimetic skeletal and cardiac systems to allow high-throughput pathophysiological studies and drug screening. This review critically analyzes microfluidic platforms that were designed for skeletal and cardiac muscle tissue engineering. Our aim is to highlight which specific features of the engineered systems promoted a typical reorganization of the engineered construct and to discuss how promising design solutions exploited for skeletal muscle models could be applied to improve cardiac tissue models and vice versa

Investigating interactions between epicardial adipose tissue and cardiac myocytes: what can we learn from different approaches?

Heart disease is a major cause of morbidity and mortality throughout the world. Some cardiovascular conditions can be modulated by lifestyle factors such as increased exercise or a healthier diet, but many require surgical or pharmacological interventions for their management. More targeted and less invasive therapies would be beneficial. Recently it has become apparent that epicardial adipose tissue plays an important role in normal and pathological cardiac function, and it is now the focus of considerable research. Epicardial adipose tissue can be studied by imaging of various kinds, and these approaches have yielded much useful information. However at a molecular level it is more difficult to study as it is relatively scarce in animal models and, for practical and ethical reasons, not always available in sufficient quantities from patients. What is needed is a robust model system in which the interactions between epicardial adipocytes and cardiac myocytes can be studied, and physiologically relevant manipulations performed. There are drawbacks to conventional culture methods, not least the difficulty of culturing both cardiac myocytes and adipocytes, each of which has special requirements. We discuss the benefits of a three-dimensional co-culture model in which in vivo interactions can be replicated

Cardiac multi-scale investigation of the right and left ventricle ex vivo: a review

The heart is a complex multi-scale system composed of components integrated at the subcellular, cellular, tissue and organ levels. The myocytes, the contractile elements of the heart, form a complex three-dimensional (3D) network which enables propagation of the electrical signal that triggers the contraction to efficiently pump blood towards the whole body. Cardiovascular diseases (CVDs), a major cause of mortality in developed countries, often lead to cardiovascular remodeling affecting cardiac structure and function at all scales, from myocytes and their surrounding collagen matrix to the 3D organization of the whole heart. As yet, there is no consensus as to how the myocytes are arranged and packed within their connective tissue matrix, nor how best to image them at multiple scales. Cardiovascular imaging is routinely used to investigate cardiac structure and function as well as for the evaluation of cardiac remodeling in CVDs. For a complete understanding of the relationship between structural remodeling and cardiac dysfunction in CVDs, multi-scale imaging approaches are necessary to achieve a detailed description of ventricular architecture along with cardiac function. In this context, ventricular architecture has been extensively studied using a wide variety of imaging techniques: ultrasound (US), optical coherence tomography (OCT), microscopy (confocal, episcopic, light sheet, polarized light), magnetic resonance imaging (MRI), micro-computed tomography (micro-CT) and, more recently, synchrotron X-ray phase contrast imaging (SR X-PCI). Each of these techniques have their own set of strengths and weaknesses, relating to sample size, preparation, resolution, 2D/3D capabilities, use of contrast agents and possibility of performing together with in vivo studies. Therefore, the combination of different imaging techniques to investigate the same sample, thus taking advantage of the strengths of each method, could help us to extract the maximum information about ventricular architecture and function. In this review, we provide an overview of available and emerging cardiovascular imaging techniques for assessing myocardial architecture ex vivo and discuss their utility in being able to quantify cardiac remodeling, in CVDs, from myocyte to whole organ

Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes.

Serine/threonine protein phosphatase 5 (PP5) is ubiquitously expressed in eukaryotic cells; however, its function in cardiomyocytes is unknown. Under basal conditions, PP5 is autoinhibited, but enzymatic activity rises upon binding of specific factors, such as the chaperone Hsp90. Here we show that PP5 binds and dephosphorylates the elastic N2B-unique sequence (N2Bus) of titin in cardiomyocytes. Using various binding and phosphorylation tests, cell-culture manipulation, and transgenic mouse hearts, we demonstrate that PP5 associates with N2Bus in vitro and in sarcomeres and is antagonistic to several protein kinases, which phosphorylate N2Bus and lower titin-based passive tension. PP5 is pathologically elevated and likely contributes to hypo-phosphorylation of N2Bus in failing human hearts. Furthermore, Hsp90-activated PP5 interacts with components of a sarcomeric, N2Bus-associated, mechanosensor complex, and blocks mitogen-activated protein-kinase signaling in this complex. Our work establishes PP5 as a compartmentalized, well-controlled phosphatase in cardiomyocytes, which regulates titin properties and kinase signaling at the myofilaments

Monitoring single heart cell biology using lab-on-a- chip technologies

Abstract There has been considerable interest in developing microsensors integrated within lab-on-a-chip structures for the analysis of single cells; however, substantially less work has focused on developing "active" assays, where the cell‘s metabolic and physiological function is itself controlled on-chip. The heart attack is considered the largest cause of mortality and morbidity in the western world. Dynamic information during metabolism from a single heart cell is difficult to obtain. There is a demand for the development of a robust and sensitive analytical system that will enable us to study dynamic metabolism at single-cell level to provide intracellular information on a single-cell scale in different metabolic conditions (such as healthy or simulated unhealthy conditions). The system would also provide medics and clinicians with a better understanding of heart disease, and even help to find new therapeutic compounds. Towards this objective, we have developed a novel platform based on five individually addressable microelectrodes, fully integrated within a microfluidic system, where the cell is electrically stimulated at pre-determined rates and real-time ionic and metabolic fluxes from active, beating single heart cells are measured. The device is comprised of one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an enzyme-modified lactate microbiosensor, used to measure the amounts of lactate produced by the heart cell. The device also enables simultaneous in-situ microscopy, allowing optical measurements of single-cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+ from the single beating heart cell at the same time, providing details of its electrical and metabolic state. Further, we have developed a robust microfluidic array, wherein a sensor array is integrated within an array of polydimethylsiloxane (PDMS) chambers, enabling the efficient manipulation of single heart cells and real-time analysis without the need to regenerate either working electrodes or reference electrodes fouled by any extracellular constituents. This sensor array also enables simultaneous electrochemical and optical measurements of single heart cells by integrating an enzyme-immobilized microsensor. Using this device, the fluorescence measurements of intracellular pH were obtained from a single beating heart cell whose electrical and metabolic states were controlled. The mechanism of released intracellular [H+] was investigated to examine extracellular pH change during contraction. In an attempt to measure lactate released from the electrically stimulated contracting cell, the cause of intracellular pH change is discussed. The preliminary investigation was made on the underlying relationship between intracellular pH and lactate from single heart cells in controlled metabolic states

The effects of aging on cardiac mechanics

It is well established that the aging heart exhibits left ventricular (LV) diastolic dysfunction and changes in mechanical properties, which have been attributed to alterations in the extracellular matrix (ECM). The investigators tested the hypothesis that the mechanical properties of cardiac myocytes significantly change with aging thereby contributing to the LV diastolic dysfunction. Cellular mechanical properties were determined by indenting cells with an atomic force microscope (AFM). The indentation results were interpreted by modeling the AFM probe as a blunted cone and determining an apparent elastic modulus (B) with classical infinitesimal strain theory (CIST). A commercially available finite element software package (ABAQUS) was used to further explore nano-indentation and the use of CIST to determine material properties. The cellular mechanical property changes, measured in young and old cardiac cells isolated from rats, showed a significant increase (p\u3c0.05) in B with aging. Cellular protein changes were assessed by immunoblot (western) analyses in order to establish if material property changes also occurred with aging. The western results indicate significant (p\u3c0.05) changes in cytoskeletal and mechanotransduction proteins with aging. These data support the concept that the mechanism mediating LV diastolic dysfunction in the aging hearts resides, in part, at the level of the myocyte. The effect of these aging induced cellular changes on global cardiac function will be further explored with instrumentation developed for implantation in an in vivo animal model

Device for Evaluating the Contraction of Cardiac Cell-Seeded Fibers

Drug-induced cardiotoxicity remains a primary reason why new pharmaceutical compounds are withdrawn from clinical trials and the consumer market. We have developed a novel cardiotoxicity screening device that utilizes cardiomyocytes seeded on fibrin microthreads to form a three-dimensional model for cardiac tissue. Our device can be used to analyze the contractile properties of these seeded threads. Upon the addition of pharmaceutical agents, the device has the potential to detect changes in contraction and thus identify cardiotoxic compounds

Modeling Cardiomyopathies in a Dish: State-of-the-Art and Novel Perspectives on hiPSC-Derived Cardiomyocytes Maturation

The stem cell technology and the induced pluripotent stem cells (iPSCs) production represent an excellent alternative tool to study cardiomyopathies, which overcome the limitations associated with primary cardiomyocytes (CMs) access and manipulation. CMs from human iPSCs (hiPSC–CMs) are genetically identical to patient primary cells of origin, with the main electrophysiological and mechanical features of CMs. The key issue to be solved is to achieve a degree of structural and functional maturity typical of adult CMs. In this perspective, we will focus on the main differences between fetal‐like hiPSC‐CMs and adult CMs. A viewpoint is given on the different approaches used to improve hiPSC‐CMs maturity, spanning from long‐term culture to complex engineered heart tissue. Further, we outline limitations and future developments needed in cardiomyopathy disease modeling.Fil: Lodola, Francesco. Università degli Studi di Milano; ItaliaFil: de Giusti, Verónica Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Maniezzi, Claudia. Università degli Studi di Milano; ItaliaFil: Martone, Daniele. Università degli Studi di Milano; ItaliaFil: Stadiotti, Ilaria. Università degli Studi di Milano; ItaliaFil: Sommariva, Elena. Università degli Studi di Milano; ItaliaFil: Maione, Angela Serena. Università degli Studi di Milano; Itali