Chemically imaging living cells by scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) is useful in probing and characterizing interfaces at high resolution. In this paper, the general principles of this technique are described and several applications of SECM to biological systems, particularly to living cells, is discussed, along with several example systems. Thiodione was detected and monitored electrochemically during the treatment of hepatocytes with cytotoxic menadione. The antimicrobial effects of silver(I) was followed by SECM through bacterial respiration. Living HeLa cells were shown to accumulate ferrocencemethanol (FcMeOH) and generated positive feedback for FcMeOH oxidation that can be further used to monitor the cell viability. Finally, individual giant liposomes, as cell models, with encapsulated redox compounds were successfully probed by SECM. In general SECM has the advantage of very high spatial resolution and versatility, especially for the detection of electroactive substances.     

Electrochemical immunoassay for CD10 antigen using scanning electrochemical microscopy

In the present study, a scanning electrochemical microscopic (SECM) method for imaging of antigen/antibody binding was proposed using CD10 antigen as the model. On the basis of anti-CD10 modified electrode, an electrochemical immunosensor for sensitive detection of CD10 antigen at low potential was developed by a multiple signal amplification strategy. Gold nanoparticles (AuNPs) served as carriers to load more secondary antibodies (Ab(2)) and horseradish peroxidase (HRP). The tip ultramicroelectrode was used to monitor the reduction current, and the 3-D images were obtained simultaneously. Under optimized conditions, the approach provided a linear response range from 1.0×l0(-11) to 6.0×l0(-11) M with a detection limit of 4.38×10(-12)M. SECM is a versatile system that can be used not only for quantitative current analysis but also for topographic imaging of binding reaction. In addition, specific binding of antigen-antibody could also be continuously and successfully monitored by SECM. This immunoassay provides a sensitive approach for detecting tumor marker, and has potential application in clinical diagnostics.     

Permeabilized rat cardiomyocyte response demonstrates intracellular origin of diffusion obstacles

Intracellular diffusion restrictions for ADP and other molecules have been predicted earlier based on experiments on permeabilized fibers or cardiomyocytes. However, it is possible that the effective diffusion distance is larger than the cell dimensions due to clumping of cells and incomplete separation of cells in fiber preparations. The aim of this work was to check whether diffusion restrictions exist inside rat cardiomyocytes or are caused by large effective diffusion distance. For that, we determined the response of oxidative phosphorylation (OxPhos) to exogenous ADP and ATP stimulation in permeabilized rat cardiomyocytes using fluorescence microscopy. The state of OxPhos was monitored via NADH and flavoprotein autofluorescence. By varying the ADP or ATP concentration in flow chamber, we determined that OxPhos has a low affinity in cardiomyocytes. The experiments were repeated in a fluorometer on cardiomyocyte suspensions leading to similar autofluorescence changes induced by ADP as recorded under the microscope. ATP stimulated OxPhos more in a fluorometer than under the microscope, which was attributed to accumulation of ADP in fluorometer chamber. By calculating the flow profile around the cell in the microscope chamber and comparing model solutions to measured data, we demonstrate that intracellular structures impose significant diffusion obstacles in rat cardiomyocytes.     

Isolation of bovine cardiomyocytes for reprogramming studies based on nuclear transfer

The goal of this study was to establish and validate a protocol for preparing bovine cardiomyocytes from slaughterhouse material for nuclear transfer experiments. The cardiomyocyte was selected because it is a terminally differentiated cell and strongly expresses a unique subset of genes which can be monitored during the reprogramming period. A total of 39 trials were conducted, and an optimized protocol was developed yielding individual contractile cardiomyocytes from 3-5-month-old bovine fetuses The basic protocol involves stabilization of bovine heart tissue for transportation from the slaughterhouse to the laboratory by perfusion with Custodiol. This was followed by an enzymatic dissociation with collagenase in calcium-free medium and yielded individual contractile rod-shaped cardiomyocytes. Subsequent addition of Ca2+ caused the cardiomyocytes to round up which was an essential pre-condition for drawing them into glass transfer pipettes for delivery into the perivitelline space and for efficient electrofusion with cytoplasts derived from in vitro matured bovine oocytes. The use of cardiomyocytes maintained at 37 degrees C in nuclear transfer, resulted in a significantly reduced proportion of blastocysts compared to adult fibroblasts (14.0% versus 32.7%). Storage of cardiomyocytes at 4 degrees C prior to nuclear transfer was not compatible with blastocyst development. It is expected that this system will be valuable for investigating the reprogramming of gene expression which occurs after somatic cell nuclear transfer.     

Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring

The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors.     

Advances in the application of scanning electrochemical microscopy to bioanalytical systems

Scanning electrochemical microscopy (SECM) is a powerful surface characterisation technique that allows for the electrochemical profiling of surfaces with sub micrometer resolution. While SECM has been most widely used to electrochemically study and profile non-biological surfaces and processes, the technique has in recent years, been increasingly used for the study of biological systems - and this is the focus of this review. An overview of SECM and how the technique may be applied to the study of biological systems will first be given. SECM and its application to the study of cells, enzymes and DNA will each be considered in detail. The review will conclude with a discussion of future directions and scope for further developments and applications.     

Real-time detection of Cu2+ sequestration and release by immobilized apo-metallothioneins using SECM combined with SPR

Scanning electrochemical microscopy (SECM) combined with surface plasmon resonance (SPR), SECM-SPR, was applied for real-time detection of the incorporation of Cu(2+) by apo-metallothionein (apo-MT) immobilized on the SPR substrate and release of Cu(2+) from surface-confined metallothionein (MT). Cu(2+) anodically stripped from a Cu-coated SECM Au tip was sequestered by apo-MT upon its diffusion to the SPR substrate, and release of Cu(2+) by MT was accomplished by generating protons via oxidation of hydroquinone at the tip. The high sensitivity of the SPR instrument is capable of following the structural and compositional changes of MT molecules during the metal sequestration and release processes. Due to the enhanced mass transfer rate at the SECM tip, the complication of mass transfer limitation on kinetic measurements, commonly encountered in flow injection SPR, is circumvented. The time-resolved SPR response reveals stepwise changes among three stable MT structures and allows the number of copper ions coordinated in each structure to be determined. The numbers of copper ions incorporated by each MT molecule in the three structures were determined to be 5, 9, and 12. This work expands the SECM-SPR approach to assessments of the dynamics and affinity of binding of small ions to surface-confined proteins and to studies of proteins that do not undergo facile electron transfer reactions.     

Photosynthetic Electron Transport in Single Guard Cells as Measured by Scanning Electrochemical Microscopy

Scanning electrochemical microscopy (SECM) is a powerful new tool for studying chemical and biological processes. It records changes in faradaic current as a microelectrode ([less than equal]7 [mu]m in diameter) is moved across the surface of a sample. The current varies as a function of both distance from the surface and the surface's chemical and electrical properties. We used SECM to examine in vivo topography and photosynthetic electron transport of individual guard cells in Tradescantia fluminensis, to our knowledge the first such analysis for an intact plant. We measured surface topography at the micrometer level and concentration profiles of O2 evolved in photosynthetic electron transport. Comparison of topography and oxygen profiles above single stomatal complexes clearly showed photosynthetic electron transport in guard cells, as indicated by induction of O2 evolution by photosynthetically active radiation. SECM is unique in its ability to measure topography and chemical fluxes, combining some of the attributes of patch clamping with scanning tunneling microscopy. In this paper we suggest several questions in plant physiology that it might address.     

"Turnover proteome" of human atrial trabeculae

Most of the biologically relevant data on cardiomyocytes are derived from isolated cells under conditions that are, to some extent, altered compared to the natural milieu of the functional heart. The handling procedure of the dissection, isolation, and short-term culturing induces changes in the cells such that the subsequently measured parameters (among others, the protein synthesis) reflect the actual experimental conduct rather than the intrinsic properties of these terminally differentiated cells. Although it is known that the protein synthetic machinery of isolated cardiomyocytes is operational and functional, the biosynthetic yield of human cardiomyocytes in the natural milieu of the trabeculae remains to be established, with a special emphasis to clarify whether the protein synthesis includes just a limited set of polypeptides or it encompasses all cellular constituents. Knowledge on this issue is a prerequisite for achieving further advances in our understanding of heart remodeling related to hypertrophy in particular, and for attempting interventions leading to repair of damaged heart in general. The experimental system of "organ bath" enables simultaneous registration of contractile forces of portions of cardiac muscle tissue (and other myocyte-containing tissues) and biosynthetic labeling of newly synthesized cellular constituents. The organ bath methodology was adapted for this project such as enabling to measure molecular changes in response to in vitro applied stimuli. Instead of Krebs-Henseleit-solution, as used in classical protocols of organ bath studies, we utilized cell culture media suitable to experimental conditions related to metabolic labeling. Proteome patterns established by performing two-dimensional gel electrophoresis of the extracts from biosynthetically labeled trabeculae revealed that cardiomyocytes synthesize the full spectrum of cellular proteins. Proteomic silver-stain readout was used to obtain samples for spot excisions, as material suitable for mass spectrometric analysis. Protein spots were identified both from the excised spots and also by matching with the in-house- and www-databases (Swiss-Prot/High-Performance Heart). From our findings that protein synthesis in terminally differentiated cardiomyocytes is not confined just to the synthesis of those structures needed for the post-mitotic house-keeping functions, we conclude that this model might serve as a valid experimental system to study and elucidate the effects of various pharmacological compounds under conditions where physiology (contractile forces) and biochemistry (protein synthesis) of intact human heart tissue are monitored simultaneously.     

Induction of premature senescence in cardiomyocytes by doxorubicin as a novel mechanism of myocardial damage

Cellular senescence is an important phenomenon in decreased cellular function. Recently, it was shown that cellular senescence is induced in proliferating cells within a short period of time by oxidative stresses. This phenomenon is known as premature senescence. However, it is still unknown whether premature senescence can be also induced in cardiomyocytes. The aim of the present study was to investigate whether a senescence-like phenotype can be induced in cardiomyocytes by oxidative stress. In cardiomyocytes obtained from aged rats (24 months of age), the staining for senescence-associated beta-galactosidase increased significantly and the protein or RNA levels of cyclin-dependent kinase inhibitors increased compared to those of young rats. Decreased cardiac troponin I phosphorylation and telomerase activity were also observed in aged cardiomyocytes. Treatment of cultured neonatal rat cardiomyocytes with a low concentration of doxorubicin (DOX) (10(-7) mol L(-1)) did not induce apoptosis but did induce oxidative stress, which was confirmed by 2',7'-dichlorofluorescin diacetate staining. In DOX-treated neonatal cardiomyocytes, increased positive staining for senescence-associated beta-galactosidase, cdk-I expression, decreased cardiac troponin I phosphorylation, and decreased telomerase activity were observed, as aged cardiomyocytes. Alterations in mRNA expression typically seen in aged cells were observed in DOX-treated neonatal cardiomyocytes. We also found that promyelocytic leukemia protein and acetylated p53, key proteins involved in stress-induced premature senescence in proliferating cells, were associated with cellular alterations of senescence in DOX-treated cardiomyocytes. In conclusion, cardiomyocytes treated with DOX showed characteristic changes similar to cardiomyocytes of aged rats. promyelocytic leukemia-related p53 acetylation may be an underlying mechanism of senescence-like alterations in cardiomyocytes. These findings indicate a novel mechanism of myocardial dysfunction induced by oxidative stress.     

Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines

Efficient differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to a variety of lineages requires step-wise approaches replicating the key commitment stages found during embryonic development. Here we show that expression of PdgfR-α segregates mouse ESC-derived Flk-1 mesoderm into Flk-1(+)PdgfR-α(+) cardiac and Flk-1(+)PdgfR-α(-) hematopoietic subpopulations. By monitoring Flk-1 and PdgfR-α expression, we found that specification of cardiac mesoderm and cardiomyocytes is determined by remarkably small changes in levels of Activin/Nodal and BMP signaling. Translation to human ESCs and iPSCs revealed that the emergence of cardiac mesoderm could also be monitored by coexpression of KDR and PDGFR-α and that this process was similarly dependent on optimal levels of Activin/Nodal and BMP signaling. Importantly, we found that individual mouse and human pluripotent stem cell lines require optimization of these signaling pathways for efficient cardiac differentiation, illustrating a principle that may well apply in other contexts.     

Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling

Optimal timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling and is mediated via PKC and NO synthase (NOS). Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells was monitored for viability and NO generation. Normothermic I/R resulted in 49.7 +/- 3.4% cell death. Hypothermia induction to 25 degrees C was most protective (14.3 +/- 0.6% death, P < 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared with induction after reperfusion (22.4 +/- 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 +/- 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase-3 activation. The NOS inhibitor N(omega)-nitro-L-arginine methyl ester (200 microM) reversed these changes and abrogated hypothermia protection. In addition, the PKCepsilon inhibitor myr-PKCepsilon v1-2 (5 microM) also reversed NO production and hypothermia protection. In conclusion, therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection appears to be mediated by increased NO generation via activation of protein kinase Cepsilon; nitric oxide synthase.     

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.     

Isolated cardiomyocytes in conjunction with NMR spectroscopy techniques to study metabolism and ion flux.

To distinguish cellular from vascular responses to physiological and pathophysiological stimuli, we developed methods to perform NMR spectroscopy on isolated ventricular cardiomyocytes. Isolated adult rat cardiomyocytes, placed in agarose beads and superfused with phosphate-free buffer (Media 199 (GIBCO 400-1100) gassed with 95% O2, 5% CO2), were used to evaluate a variety of cellular processes during different pharmacological and physiological interventions. Bioenergetic function was monitored with 31P NMR. Intermediary metabolism, gluconeogenesis, and glycolysis were monitored with 13C NMR. Sodium flux was monitored with 23Na NMR. Calcium flux was monitored with 19F NMR in conjunction with an intracellular calcium-chelating agent, 5F-1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid. Creatine kinase kinetics (forward rate constant (Kf) and flux of phosphocreatine to ATP) were estimated with 31P NMR saturation transfer data. Various combinations of NMR parameters were monitored simultaneously so that the interaction of metabolism and ion flux could be evaluated. We have demonstrated that it is possible to simultaneously monitor a variety of cellular processes in intact heart cells in real time, without the confounding influences of perfusion, contractile function, and extrinsic blood-borne neurohumoral agents. This model will be useful for longitudinal studies of myocyte metabolism and ion flux.     

Investigation of concentration profiles inside operating biocatalytic sensors with scanning electrochemical microscopy (SECM)

Scanning electrochemical microscopy (SECM) with amperometric or potentiometric measuring tips was used to investigate biocatalytic reactions inside the enzyme layer of a biosensor during its operation. The well known glucose oxidase catalyzed oxidation of glucose has been selected for the studies. Local, instantaneous concentration of dissolved oxygen and hydrogen peroxide was studied observing the amperometric current while miniaturized potentiometric tip served for local pH measurements. Liquid enzyme layer immobilized with Cellophane membrane or cross linked polyacrilamide gel membrane containing entrapped enzyme served for biocatalytic media in the SECM imaging. Local maximum of H(2)O(2) and minimum of O(2) profiles were found at approximately 200 microm far from the substrate/enzyme layer boundary. From the experimental findings guidelines to design well functioning biocatalytic sensors could be concluded. The concentration profiles obtained with SECM techniques were compared with the results of simple model calculations carried out with the method of finite changes. Most of earlier made SECM studies dealing with enzyme reactions imaged the electrolyte being in contact with the immobilized enzyme. The data in our investigation, however, were collected inside the working catalytic layer.     

HSP90 and eNOS partially co-localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes

Background information. Cultivation techniques promoting three‐dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel‐based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co‐cultures to evaluate, on both two‐ and three‐dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat‐shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). Results. Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co‐culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel‐cultured cardiomyocytes, areas of high co‐localization between HSP90 and eNOS and co‐localization coefficients, which indicated the highest correlation with respect to the other substrates. Conclusions. Our three‐dimensional adult cardiomyocyte cultures are suitable for both analysing cell—ECM interactions at electron and confocal microscopy levels and monitoring micro‐environment impact on cardiomyocyte phenotype.     

An Optimized Protocol for Culture of Cardiomyocyte from Neonatal Rat

Primary culture of cardiomyocytes has been widely used as a valuable tool for pharmacological and toxicological studies. However, the fact that heart is a solid organ and cardiomyocytes do not proliferate after birth makes the primary myocardial culture a tedious job. The present study reports an improved method for rapid isolation of cardiomyocytes, as well as the culture maintenance and quality assurance. The whole culture process can be shortened to 3.5 h by reducing enzyme digestion period. Moreover, the new protocol guarantees cell yield and viability, and produces more than 95% cardiomyocytes in culture. The cardiomyocytes can respond to Angiotension II stimulation with increased protein synthesis, suggesting the practical value of this new culture method.     

The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin

In failing hearts cardiomyocytes undergo alterations in cytoskeleton structure, contractility and viability. It is not known presently, how stress-induced changes of myofibrils correlate with markers for cell death and contractile function in cardiomyocytes. Therefore, we have studied the progression of contractile dysfunction, myofibrillar damage and cell death in cultured adult cardiomyocytes exposed to the cancer therapy doxorubicin. We demonstrate, that long-term cultured adult cardiomyocytes, a well-established model for the study of myofibrillar structure and effects of growth factors, can also be used to assess contractility and calcium handling. Adult rat ventricular myocytes (ARVM) were isolated and cultured for a total of 14 days in serum containing medium. The organization of calcium-handling proteins and myofibrillar structure in freshly isolated and in long-term cultured adult cardiomyocytes was studied by immunofluorescence and electron microscopy. Excitation contraction-coupling was analyzed by fura 2 and video edge detection in electrically paced cardiomyocytes forming a monolayer, and cell death and viability was measured by TUNEL assay, LDH release, MTT assay, and Western blot for LC3. Adult cardiomyocytes treated with Doxo showed apoptosis and necrosis only at supraclinical concentrations. Treated cells displayed merely alterations in cytoskeleton organization and integrity concomitant with contractile dysfunction and up-regulation of autophagosome formation, but no change in total sarcomeric protein content. We propose, that myofibrillar damage contributes to contractile dysfunction prior to cell death in adult cardiomyocytes exposed to clinically relevant concentrations of anthracyclines.