Beating Heart Cells Research Articles

Beating heart cells: Using cultured cardiomyocytes to study cellular structure and contractility in laboratory exercises.

Heart muscle cells, or cardiomyocytes, exhibit intrinsic contractility in vitro. We found that commercially-available mammalian cardiomyocytes serve as an excellent model system for studying the cytoskeleton and cellular contractility, fundamental topics in undergraduate cell and molecular biology courses. Embryonic rat cardiomyocytes were plated on cell culture dishes or glass coverslips and visualized using an inverted phase-contrast microscope. The cardiomyocytes began contracting within 1-2 days after plating and continued to contract for many weeks, allowing their use in multiple laboratory sessions. Following background reading and instruction, students fixed and triple-stained the cardiomyocytes to examine the relative distributions of actin filaments and microtubules and the position of nuclei. Analysis and image capture with fluorescence microscopy provided striking examples of highly organized cytoskeletal elements. Students then designed experiments in which cardiomyocyte intrinsic contractility was explored. Changes in contraction rates were examined after treatment with signaling molecules, such as epinephrine. The addition of epinephrine to the culture medium, within a usable concentration window, increased the rate of contraction. These adaptable exercises provide undergraduate cell and molecular biology students with the exciting opportunity to study cardiomyocytes using standard cell culture and microscopy techniques.

Double explosive transitions to synchronization and cooperation in intertwined dynamics and evolutionary games

Collective behavior, from murmurations to synchronized beating of heart cells, governs some of the most beautiful and important aspects of nature. Likewise, cooperation—the act of sacrificing personal benefits for the common good—is one of the pillars of social evolution, and it is the basis for the emergence of collective organized actions from single-cell organisms to modern human societies. Here we merge these two phenomena into a single model, considering an ensemble of networked oscillators, where each oscillator can be either a cooperator or a defector, and with only cooperators contributing to synchrony. At the same time, the value of the order parameter in the neighborhood of each oscillator is considered as an effective local temperature which determines the strategy updating procedure in the evolutionary game. The emergence of cooperation is thus intertwined with that of synchronization, producing a novel and fascinating dynamics which includes a double explosive transition.

Dynamic localization of αB-crystallin at the microtubule cytoskeleton network in beating heart cells.

αB-crystallin is highly expressed in the heart and slow skeletal muscle; however, the roles of αB-crystallin in the muscle are obscure. Previously, we showed that αB-crystallin localizes at the sarcomere Z-bands, corresponding to the focal adhesions of cultured cells. In myoblast cells, αB-crystallin completely colocalizes with microtubules and maintains cell shape and adhesion. In this study, we show that in beating cardiomyocytes α-tubulin and αB-crystallin colocalize at the I- and Z-bands of the myocardium, where it may function as a molecular chaperone for tubulin/microtubules. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the striated patterns of GFP-αB-crystallin fluorescence recovered quickly at 37°C. FRAP mobility assay also showed αB-crystallin to be associated with nocodazole-treated free tubulin dimers but not with taxol-treated microtubules. The interaction of αB-crystallin and free tubulin was further confirmed by immunoprecipitation and microtubule sedimentation assay in the presence of 1-100 μM calcium, which destabilizes microtubules. Förster resonance energy transfer analysis showed that αB-crystallin and tubulin were at 1-10 nm apart from each other in the presence of colchicine. These results suggested that αB-crystallin may play an essential role in microtubule dynamics by maintaining free tubulin in striated muscles, such as the soleus or cardiac muscles.

Implications for the Predictivity of Cell-Based Developmental Toxicity Assays Developed Two Decades Apart

Many in vitro developmental toxicity assays have been proposed over several decades. Since the late 1980s, we have made intermittent attempts to introduce in vitro assays as screening tests for developmental toxicity of in-house candidate products. Two cell-based assays which were developed two decades apart were intensively studied. One was an assay of inhibitory effects on mouse ascites tumor cell attachment to a concanavalin A-coated plastic sheet surface (MOT assay), which we studied in the early days of assay development. The other was an assay of inhibitory effects on the differentiation of mouse embryonic stem cell to beating heart cells (EST assay), which we assessed more recently. We evaluated the suitability of the assays for screening in-house candidates. The concordance rates with in vivo developmental toxicity were at the 60% level. The EST assay classified chemicals that inhibited cell proliferation as embryo-toxic. Both assays had a significant false positive rate. The assays were generally considered unsuitable for screening the developmental toxicity of our candidate compounds. Recent test systems adopt advanced technologies. Despite such evolution of materials and methods, the concordance rates of the EST and MOT systems were similar. This may suggest that the fundamental predictivity of in vitro developmental toxicity assays has remained basically unchanged for decades. To improve their predictivity, in vitro developmental toxicity assays should be strictly based on elucidated pathogenetic mechanisms of developmental toxicity.

Living Matter: Mesoscopic Active Materials

An introduction to the physical properties of living active matter at the mesoscopic scale (tens of nanometers to micrometers) and their unique features compared with "dead," nonactive matter is presented. This field of research is increasingly denoted as "biological physics" where physics includes chemical physics, soft matter physics, hydrodynamics, mechanics, and the related engineering sciences. The focus is on the emergent properties of these systems and their collective behavior, which results in active self-organization and how they relate to cellular-level biological function. These include locomotion (cell motility and migration) forces that give rise to cell division, the growth and form of cellular assemblies in development, the beating of heart cells, and the effects of mechanical perturbations such as shear flow (in the bloodstream) or adhesion to other cells or tissues. An introduction to the fundamental concepts and theory with selected experimental examples related to the authors' own research is presented, including red-blood-cell membrane fluctuations, motion of the nucleus within an egg cell, self-contracting acto-myosin gels, and structure and beating of heart cells (cardiomyocytes), including how they can be driven by an oscillating, mechanical probe.

Nanofibre optic force transducers with sub-piconewton resolution via near-field plasmon-dielectric interactions.

Ultrasensitive nanomechanical instruments, including the atomic force microscope (AFM)1-4 and optical and magnetic tweezers5-8, have helped shed new light on the complex mechanical environments of biological processes. However, it is difficult to scale down the size of these instruments due to their feedback mechanisms9, which, if overcome, would enable high-density nanomechanical probing inside materials. A variety of molecular force probes including mechanophores10, quantum dots11, fluorescent pairs12,13 and molecular rotors14-16 have been designed to measure intracellular stresses; however, fluorescence-based techniques can have short operating times due to photo-instability and it is still challenging to quantify the forces with high spatial and mechanical resolution. Here, we develop a compact nanofibre optic force transducer (NOFT) that utilizes strong near-field plasmon-dielectric interactions to measure local forces with a sensitivity of <200 fN. The NOFT system is tested by monitoring bacterial motion and heart-cell beating as well as detecting infrasound power in solution.

Identification of cardiac progenitors that survive in the ischemic human heart after ventricular myocyte death

Atypically-shaped cardiomyocytes (ACMs) are beating heart cells identified in the cultures of cardiomyocyte-removed fractions obtained from adult mouse hearts. Since ACMs spontaneously develop into beating cells in the absence of hormones or chemicals, these cells are likely to be a type of cardiac progenitors rather than stem cells. “Native ACMs” are found as small interstitial cells among ventricular myocytes that co-express cellular prion protein (PrP) and cardiac troponin T (cTnT) in mouse and human heart tissues. However, the endogenous behavior of human ACMs is unclear. In the present study, we demonstrate that PrP+ cTnT+ cells are present in the human heart tissue with myocardial infarction (MI). These cells were mainly found in the border of necrotic cardiomyocytes caused by infarcts and also in the hibernating myocardium subjected to the chronic ischemia. The ratio of PrP+ cTnT+ cells to the total cells observed in the normal heart tissue section of mouse and human was estimated to range from 0.3–0.8%. Notably, living human PrP+ cTnT+ cells were identified in the cultures obtained at pathological autopsy despite exposure to lethal ischemic conditions for hours after death. These findings suggest that ACMs could survive in the ischemic human heart and develop into a sub-population of cardiac myocytes.

Substrate Regulation of Cytoskeletal Order and Beating Strain Dynamics of Cardiomyocytes

Recent experiments [1] show that both striation, an indication of the structural registry in muscle fibers, as well as the contractile strains produced by beating cardiac muscle cells, can be optimized by substrate stiffness. We show theoretically how the substrate rigidity dependence of the registry data can be mapped onto that of the strain measurements. We determine [2] the elasticity-mediated structural registry including both the elastic interactions of neighboring, actively contractile acto-myosin units as well as the noise inherent in biological systems. By assuming that structurally registered myofibrils also tend to beat in phase, we explain the observed dependence of both structural striation and beating strain measurements of heart muscle cells on substrate stiffness in a unified manner. The agreement of our ideas with experiment suggests that the correlated beating of heart cells may be limited by the structural registry of the myofibrils which in turn is regulated by their elastic environment. New theoretical predictions focus on the role of the substrate and the intercellular distance in the synchronization of the beating of cellular assemblies. [1] S. Majkut et al., Current Biology, 23, 2323 (2013). [2] K. Dasbiswas, S. Majkut, D. Discher and S. Safran, Nature Comm., 6, 7085 (2015). Experimental Collaborations - Dennis Discher, Stephanie Majkut (Penn); Shelly Tzlil (Technion).

Joseph C. Wu: Envisioning Personalized Medicine and Clinical Trials in a Dish.

If he develops heart disease in the future, Joseph Wu envisions his physicians selecting the ideal medication for him by first testing drugs on his “mini-me” surrogates – dishes of beating heart cells created from his own stem cells. Joseph C. Wu Growing up in Southern California, Wu helped his father grow Asian specialty pears. Now, as director of the Stanford Cardiovascular Institute in Palo Alto, California, he grows cardiomyocytes for a wide range of applications – disease modeling, drug discovery, medication safety testing, and possible regenerative therapy. Since coming to Stanford in 2004, Wu has worked with both human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells. Using ES cells, Dr Wu’s laboratory is addressing several important technical challenges in stem cell technology, including how to improve cardiac differentiation efficiency,1 control potential tumorigenicity,2 and avoid immune rejection.3 For iPS cells, his laboratory has shown that iPS cell-derived cardiomyocytes can be used to elucidate the molecular mechanisms of familial dilated cardiomyopathy and hypertrophic cardiomyopathy.4–6 In a paper designated the best basic science manuscript in Circulation in 2014, Wu used a library of iPS cell-derived cardiomyocytes to show that patterns of drug-induced cardiotoxicity differed among healthy controls and patients with long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy, indicating that the system might be a more accurate predictor of adverse drug responses than current assays and screening tests.7 In Circulation Research , he recently described the opportunities to use iPS cell-derived cardiomyocytes to advance the scientific understanding and treatment of sudden cardiac death.8 The Stanford Cardiovascular Institute is currently creating a biobank of patient-specific, deidentified iPS cell lines from healthy controls and patients with a wide range of cardiovascular diseases, which will benefit the research community and patients alike.9 …

A beating heart cell model to predict cardiotoxicity: Effects of the dietary supplement ingredients higenamine, phenylethylamine, ephedrine and caffeine

Some dietary supplements may contain cardiac stimulants and potential cardiotoxins. In vitro studies may identify ingredients of concern. A beating human cardiomyocyte cell line was used to evaluate cellular effects following phenylethylamine (PEA), higenamine, ephedrine or caffeine treatment. PEA and higenamine exposure levels simulated published blood levels in humans or animals after intravenous administration. Ephedrine and caffeine levels approximated published blood levels following human oral intake. At low or midrange levels, each chemical was examined plus or minus 50 µM caffeine, simulating human blood levels reported after consumption of caffeine-enriched dietary supplements. To measure beats per minute (BPM), peak width, etc., rhythmic rise and fall in intracellular calcium levels following 30 min of treatment was examined. Higenamine 31.3 ng/ml or 313 ng/ml significantly increased BPM in an escalating manner. PEA increased BPM at 0.8 and 8 µg/ml, while 80 µg/ml PEA reduced BPM and widened peaks. Ephedrine produced a significant BPM dose response from 0.5 to 5.0 µM. Caffeine increased BPM only at a toxic level of 250 µM. Adding caffeine to PEA or higenamine but not ephedrine further increased BPM. These in vitro results suggest that additional testing may be warranted in vivo to further evaluate these effects.

Substrate stiffness-modulated registry phase correlations in cardiomyocytes map structural order to coherent beating

Recent experiments show that both striation, an indication of the structural registry in muscle fibres, as well as the contractile strains produced by beating cardiac muscle cells can be optimized by substrate stiffness. Here we show theoretically how the substrate rigidity dependence of the registry data can be mapped onto that of the strain measurements. We express the elasticity-mediated structural registry as a phase-order parameter using a statistical physics approach that takes the noise and disorder inherent in biological systems into account. By assuming that structurally registered myofibrils also tend to beat in phase, we explain the observed dependence of both striation and strain measurements of cardiomyocytes on substrate stiffness in a unified manner. The agreement of our ideas with experiment suggests that the correlated beating of heart cells may be limited by the structural order of the myofibrils, which in turn is regulated by their elastic environment.

Prion protein- and cardiac troponin T-marked interstitial cells from the adult myocardium spontaneously develop into beating cardiomyocytes.

Atypically-shaped cardiomyocytes (ACMs) constitute a novel subpopulation of beating heart cells found in the cultures of cardiac myocyte-removed crude fraction cells obtained from adult mouse cardiac ventricles. Although ~500 beating ACMs are observed under microscope in the cell cultures obtained from the hearts of either male or female mice, the origin of these cells in cardiac tissue has yet to be elucidated due to the lack of exclusive markers. In the present study, we demonstrate the efficacy of cellular prion protein (PrP) as a surface marker of ACMs. Cells expressing PrP at the plasma membrane in the culture of the crude fraction cells were found to develop into beating ACMs by themselves or fuse with each other to become larger multinuclear beating ACMs. Combining PrP with a cardiac-specific contractile protein cardiac troponin T (cTnT) allowed us to identify native ACMs in the mouse cardiac ventricles as either clustered or solitary cells. PrP- and cTnT-marked cells were also found in the adult, even aged, human cardiac ventricles. These findings suggest that interstitial cells marked by PrP and cTnT, native ACMs, exhibit life-long survival in the cardiac ventricles of both mice and humans.

Cardiomyocyte subdomain contractility arising from microenvironmental stiffness and topography.

Cellular structure and function are interdependent. To understand this relationship in beating heart cells, individual neonatal rat ventricular myocytes (NRVMs) were analyzed one and 3days after plating when cultured on different stiffness (100, 400kPa) and surface structures (flat or [Formula: see text] high, [Formula: see text] diameter, microposts spaced [Formula: see text] apart) manufactured from polydimethylsiloxane. Myofibril structure seen by immunohistochemistry was organized in three dimensions when NRVMs were attached to microposts. On day three, paxillin distribution near the post serving as cellular anchorage was quantified on both soft posts (12.04% of total voxel count) and stiff posts (8.16%). Living NRVMs were analyzed using line scans for sarcomeric shortening and shortening velocity, and traction force microscopy for surface stress and surface tension. One day after plating, NRVMs shortened more on soft posts ([Formula: see text] at [Formula: see text]) compared to either soft flat ([Formula: see text] at [Formula: see text]), stiff posts ([Formula: see text] at [Formula: see text]) or stiff flat ([Formula: see text] at [Formula: see text]). NRVMs have decreased shortening and shortening velocity on soft posts ([Formula: see text] at [Formula: see text]) compared to soft flat ([Formula: see text] at [Formula: see text]) substrates. The surface stress and surface tension increased over time for both soft post ([Formula: see text] and [Formula: see text] to [Formula: see text] and [Formula: see text]) and flat ([Formula: see text] and [Formula: see text] to [Formula: see text] and [Formula: see text]) substrates. Paxillin displacement during contraction on day three was significantly greater in NRVMs attached to soft posts [Formula: see text] compared to flat [Formula: see text] substrates. The volume and time creating four-dimensional data, interpreted by structural engineering theory, demonstrate subdomain structure is maintained by the counterbalance between the external load acting upon and the internal forces generated by the cardiomyocyte. These findings provide further insight into localized regulation of cellular mechanical function.

A collagen membrane-based engineered heart tissue improves cardiac function in ischemic rat hearts

In the relatively new field of cardiac tissue engineering, different biomaterials, methods and techniques have been tested for cardiac repair, but we are still far from the achievement of a valid model that can be tested for therapeutic goals. In this study, the authors examined the efficacy of newly preformed membranes based on collagen type I for the transplantation of cardiac cells. The membrane prototype, cross-linked with 1,4-butanediol diglycidyl ether (BDDGE) and fibronectin-enriched, gave rise to spontaneously beating heart cell constructs, 5–9 days after seeding with neonatal rat cardiac cells. This membrane was grafted, with and without beating cardiac cells, onto the infarcted area of rat models of heart failure. Seriate echocardiography, performed on rats before transplantation and at 4 and 8 weeks after transplantation, demonstrated that rats treated with collagen membranes previously seeded with beating cells showed an improvement in cardiac function after 8 weeks. These results suggest that this new type of collagen membrane can be used as vector for the transplantation of beating heart cells for the regeneration of the injured myocardium and hence represents an important potential tool for cardiac tissue repair technologies.

Ischemic survival and constitutively active autophagy in self-beating atypically-shaped cardiomyocytes (ACMs): characterization of a new subpopulation of heart cells

Atypically-shaped cardiomyocytes (ACMs) are a new subpopulation of spontaneously beating heart cells with a peculiar morphology identified within a culture of cardiac myocyte-depleted fraction (CMDF) cells obtained from adult mouse heart. ACMs originate from small cells in CMDF and grow in size and start beating within ~3 days culture without appreciable proliferation or express stem cell marker proteins, but stay in the heart until elderly stages. However, the characteristics of ACMs are largely unclear. The present study examined whether pre-exposure of CMDF cells to severe ischemia abolished the ability of ACMs to develop into beating cells. Of ACMs that underwent ischemia, ~50 % grew in size, changed the morphology, and started beating during the subsequent culture under normoxia. ACMs displayed constitutively active autophagy during the culture. The results suggest the possibility that the development of beating ACMs could occur in injured heart, even if the surviving cell population is small.

Videotape Illustration of Cultured Beating Heart Cells and The Application of Such Cultures to Studying The Cardiotoxicity of Adriamycin

Videotape Illustration of Cultured Beating Heart Cells and The Application of Such Cultures to Studying The Cardiotoxicity of Adriamycin

A method of preparing beating heart cells for tissue culture [proceedings

A method of preparing beating heart cells for tissue culture [proceedings

Reduction of the Toxicity of Adriamycin to Beating Heart Cells by Free-Radical Scavengers

Reduction of the Toxicity of Adriamycin to Beating Heart Cells by Free-Radical Scavengers

Microfluidic cell arrays for metabolic monitoring of stimulated cardiomyocytes

An array of PDMS microchambers was aligned to an array of sensor electrodes and stimulating microelectrodes, which was used for the electrochemical monitoring of the metabolic activity of single isolated adult ventricular myocytes inside the chamber array, stimulated within a transient electric field. The effect of the accumulation of metabolic byproducts in the limited extracellular volume of the picolitre chambers was demonstrated by measuring single muscle cell contraction optically, while concomitant changes in intracellular calcium transients and pH were recorded independently using fluorescent indicator dyes. Both the amplitude of the cell shortening and the magnitude of the intracellular calcium transients decreased over time and both nearly ceased after 20 min of continuous stimulation in the limited extracellullar volume. The intracellular pH decreased gradually during 20 min of continuous stimulation after which a dramatic pH drop was observed, indicating the breakdown of the intracellular buffering capacity. After continuous stimulation, intracellular lactate was released into the microchamber through cell electroporation and was detected electrochemically at a lactate microbiosensor, within the chamber. A mitochondrial uncoupler was used to mimic ischaemia and thus to enhance the cellular content of lactate. Under these circumstances, intracellular lactate concentrations were found to have risen to approximately 15 mM. This array system has the potential of simultaneous electrochemical and optical monitoring of extracellular and intracellular metabolites from single beating heart cells at a controlled metabolic state.

N-Myristoyltransferase 1 Is Essential in Early Mouse Development

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a beta-galactosidase marker gene. beta-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1+/-) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1+/- mice yielded no viable homozygotes (Nmt1-/-), and heterozygotes were born at a less than predicted frequency. Nmt1-/- embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1-/- embryos. To explore this concept, we generated Nmt1-/- embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1-/- ES cells, but the total NMT activity levels were reduced by approximately 95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1-/- ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis.