cultures to be used in various analytical investigations of cardiomyocyte (CM) growth and function for enhancing insight into physiological and pathological mechanisms should closely express morphology. The aim of the studies is to explore how to use microfabrication and physical-cue-addition techniques to establish a neonatal rat CM culture model that expresses an end-to-end connected rod shape with -like intercalated discs (ICDs). Freshly isolated neonatal rat CMs were cultured on microgrooved polydimethylsiloxane substrate.
View Article and Find Full Text PDFDetecting the structural changes caused by volume and pressure overload is critical to comprehending the mechanisms of physiologic and pathologic hypertrophy. This study explores the structural changes at the crystallographic level in myosin filaments in volume- and pressure-overloaded myocardia through polarization-dependent second harmonic generation microscopy. Here, for the first time, we report that the ratio of nonlinear susceptibility tensor components increased significantly in volume- and pressure-overloaded myocardial tissues compared with the ratio in normal mouse myocardial tissues.
View Article and Find Full Text PDFMyofibrils are the main protein structures that generate force in the beating heart. Myofibril disassembly is related to many physiological and pathological processes. This study investigated, in a cultured rat adult cardiomyocyte model, the effect of force imbalance on myofibril disassembly.
View Article and Find Full Text PDFAn increase in mechanical load in the heart causes cardiac hypertrophy, either physiologically (heart development, exercise and pregnancy) or pathologically (high blood pressure and heart-valve regurgitation). Understanding cardiac hypertrophy is critical to comprehending the mechanisms of heart development and treatment of heart disease. However, the major molecular event that occurs during physiological or pathological hypertrophy is the dynamic process of sarcomeric addition, and it has not been observed.
View Article and Find Full Text PDFTunneling nanotubes (TNTs) are small membranous tubes of 50-1000 nm diameter observed to connect cells in culture. Transfer of subcellular organelles through TNTs was observed in vitro and in vivo, but the formation and significance of these structures is not well understood. A polydimethylsiloxane biochip-based coculture model was devised to constrain TNT orientation and explore both TNT-formation and TNT-mediated mitochondrial transfer.
View Article and Find Full Text PDFThe current inability to derive mature cardiomyocytes from human pluripotent stem cells has been the limiting step for transitioning this powerful technology into clinical therapies. To address this, scaffold-based tissue engineering approaches have been utilized to mimic heart development in vitro and promote maturation of cardiomyocytes derived from human pluripotent stem cells. While scaffolds can provide 3D microenvironments, current scaffolds lack the matched physical/chemical/biological properties of native extracellular environments.
View Article and Find Full Text PDFProc SPIE Int Soc Opt Eng
February 2014
Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al.
View Article and Find Full Text PDFAm J Physiol Heart Circ Physiol
May 2015
Cardiac tissue slices are becoming increasingly popular as a model system for cardiac electrophysiology and pharmacology research and development. Here, we describe in detail the preparation, handling, and optical mapping of transmembrane potential and intracellular free calcium concentration transients (CaT) in ventricular tissue slices from guinea pigs and rabbits. Slices cut in the epicardium-tangential plane contained well-aligned in-slice myocardial cell strands ("fibers") in subepicardial and midmyocardial sections.
View Article and Find Full Text PDFThe basement membrane (BM), a network of laminin and collagen IV, mechanically supports individual cells and directly mediates cell-cell and cell-extracellular matrix (ECM) interactions. For example, the BM network that tightly encloses each cardiomyocyte (CM) mediates the alignment of CMs with collagen I in the ECM. Additionally, the BM-laminin is involved in the formation of gap junctions (GJs), which regulate electrical coupling between two CMs in the myocardium.
View Article and Find Full Text PDFThe cardiac basement membrane (BM), the highly organized layer of the extracellular matrix (ECM) on the external side of the sarcolemma, is mainly composed of laminin and collagen IV, which assemble a dense, well-organized network to surround the surface of each adult cardiomyocyte. The development of the cardiac BM plays a key role in organogenesis of the myocardium through interactions between sarcomeres and integrins. Because of the complicated structure of cardiac muscle fibers and lack of a proper investigation method, the detailed interactions among BM development, sarcomeric growth, and integrin expression remain unclear.
View Article and Find Full Text PDFIn the heart muscle, each adult cardiomyocyte is enclosed by a basement membrane (BM). This innermost extracellular matrix is a layered assembly of laminin, collagen IV, glycoproteins, and proteoglycans. In this study, the role of the BM network in regulation of the electrical properties of neonatal cardiomyocytes (NCMs) cultured on an aligned collagen I gel was investigated using a multielectrode array (MEA).
View Article and Find Full Text PDFA technique to tailor-make pre-coated, pre-aligned bovine collagen fibrils, derived from neonatal cardiomyocytes, on the surface of a glass slide into a designated pattern is reported. The unwanted collagen-coated area was erased by a collagenase solution and the tailored area was retained by attaching a microfabricated polydimethylsiloxane stamp directly to the collagen-coated surface. Using this technique, collagen patterns with designated orientations and with clear pattern boundaries and defined shapes were fabricated.
View Article and Find Full Text PDFMesenchymal stem cells (MSCs) have been cited as contributors to heart repair through cardiogenic differentiation and multiple cellular interactions, including the paracrine effect, cell fusion, and mechanical and electrical couplings. Due to heart-muscle complexity, progress in the development of knowledge concerning the role of MSCs in cardiac repair is heavily based on MSC-cardiomyocyte coculture. In conventional coculture systems, however, the cardiac muscle structure, in which rod-shaped cells are connected end-to-end, is not sustained; instead, irregularly shaped cells spread randomly, resulting in randomly distributed cell junctions.
View Article and Find Full Text PDFFibrillar collagen is the primary component of the cardiac interstitial extracellular matrix. This extracellular matrix undergoes dramatic changes from birth to adulthood and then into advanced age. As evidence, fibrillar collagen content was compared in sections from neonates, adult, and old hearts and was found to increase at each respective age.
View Article and Find Full Text PDFDetailed methods are provided for the preparation and confocal imaging of cardiac myocyte development and differentiation. Examples include protocols for the analysis of cultured myocytes as well as vibratome sections of hearts from embryonic and adult tissue. Techniques include routine labeling of F-actin with phalloidin as well as multiple labeling protocols for colocalization studies and cell volume analysis.
View Article and Find Full Text PDFUnderstanding how stem cells interact with cardiomyocytes is crucial for cell-based therapies to restore the cardiomyocyte loss that occurs during myocardial infarction and other cardiac diseases. It has been thought that functional myocardial repair and regeneration could be regulated by stem cell-cardiomyocyte contact. However, because various contact modes (junction formation, cell fusion, partial cell fusion, and tunneling nanotube formation) occur randomly in a conventional coculture system, the particular regulation corresponding to a specific contact mode could not be analyzed.
View Article and Find Full Text PDFNormal cardiomyocytes are highly dependent on the functional expression of ion channels to form action potentials and electrical coupling with other cells. To fully determine the scientific and therapeutic potential of stem cells for cardiovascular-disease treatment, it is necessary to assess comprehensively the regulation of stem-cell electrical properties during stem cell-cardiomyocyte interaction. It has been reported in the literature that contact with native cardiomyocytes induced and regulated stem-cell cardiogenic differentiation.
View Article and Find Full Text PDFAims: Understanding myofibrillogenesis is essential for elucidating heart muscle formation, development, and remodelling in response to physiological stimulation. Here, we report the dynamic assembly process of contractile myosin filaments onto myofibrils in a live cardiomyocyte culture during myofibrillogenesis.
Methods And Results: Utilizing a custom-built, two-photon excitation fluorescence and second harmonic generation imaging system equipped with an on-stage incubator, we observed new sarcomere additions in rat neonatal cardiomyocytes during 10 h of on-stage incubation.
Cardiac hypertrophy, whether pathological or physiological, induces a variety of additional morphological and physiological changes in the heart, including altered contractility and hemodynamics. Events exacerbating these changes are documented during later stages of hypertrophy (usually termed pathological hypertrophy). Few studies document the morphological and physiological changes during early physiological hypertrophy.
View Article and Find Full Text PDFFollowing myocardial infarction there is an irreversible loss of cardiomyocytes that results in the alteration of electrical propagation in the heart. Restoration of functional electrical properties of the damaged heart muscle is essential to recover from the infarction. While there are a few reports that demonstrate that fibroblasts can form junctions that transmit electrical signals, a potential alternative using the injection of stem cells has emerged as a promising cellular therapy; however, stem-cell electrical conductivity within the cardiac muscle fiber is unknown.
View Article and Find Full Text PDFNormal cardiac function is maintained through dynamic interactions of cardiac cells with each other and with the extracellular matrix. These interactions are important for remodeling during cardiac growth and pathophysiological conditions. However, the precise mechanisms of these interactions remain unclear.
View Article and Find Full Text PDFCell patterning methods enable researchers to control specific homotypic and heterotypic contact-mediated cell-cell and cell-ECM interactions and to impose defined cell and tissue geometries. To micropattern individual cells to specific points on a substrate with high spatial resolution, we have developed a cell deposition microscope based on the laser-guidance technique. We discuss the theory of optical forces for generating laser guidance and the optimization of the optical configuration (NA ≈ 0.
View Article and Find Full Text PDFThe heart is composed of both cellular and acellular components that act in a dynamic fashion from birth to death. The cellular components consist of myocytes, fibroblasts, and vascular cells, including endothelium and smooth muscle. Changes in these components are intimately associated with function by altering the mechanical, chemical, and electrical properties of the heart.
View Article and Find Full Text PDF