Effects of cyclosporin A on contractile activity and cytoskeleton in chick embryo cardiomyocytes.

The effects of cyclosporin A (CsA), a clinically used immunosupressive drug, on contractile activity of chick cardiomyocytes grown as small aggregates or explants suspended on a network of elastic glass fibres or cultured in a monolayer were analysed in vitro with computer-aided image cytometry methods. At therapeutic concentrations (200-1500 ng/mL), CsA induced changes in the frequency and amplitude of the beating activity of cardiomyocytes 15 min after application. Longer treatment of cardiomyocytes, for 20-24 h, additionally induced changes in their shape and cytoskeleton organization (F-actin and alpha-actinin distribution). These results indicate that CsA is able to affect directly the contractile activity, morphology, and cytoskeleton architecture of heart cells.     

Probing the role of septins in cardiomyocytes

Heart growth in the embryo is achieved by division of differentiated cardiomyocytes. Around birth, cardiomyocytes stop dividing and heart growth occurs only by volume increase of the individual cells. Cardiomyocytes seem to lose their capacity for cytokinesis at this developmental stage. Septins are GTP-binding proteins that have been shown to be involved in cytokinesis from yeast to vertebrates. We wanted to determine whether septin expression patterns can be correlated to the cessation of cytokinesis during heart development. We found significant levels of expression only for SEPT2, SEPT6, SEPT7 and SEPT9 in heart, in a developmentally regulated fashion, with high levels in the embryonic heart, downregulation around birth and no detectable expression in the adult. In dividing embryonic cardiomyocytes, all septins localize to the cleavage furrow. We used drugs to probe for the functional interactions of SEPT2 in dividing embryonic cardiomyocytes. Differences in the effects on subcellular septin localization in cardiomyocytes were observed, depending whether a Rho kinase (ROCK) inhibitor was used or whether actin and myosin were targeted directly. Our data show a tight correlation of high levels of septin expression and the ability to undergo cytokinesis in cardiomyocytes. In addition, we were able to dissect the different contributions of ROCK signaling and the actomyosin cytoskeleton to septin localization to the contractile ring using cardiomyocytes as an experimental system.     

Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy

The contractile behavior of cardiomyocytes can be monitored by measuring their action potentials, and the analysis is essential for screening the safety of potential drugs. However, immobilizing cardiac cells on a specific electrode is considerably complicated. In this study, we demonstrate that scanning electrochemical microscopy (SECM) can be used to analyze rapid topographic changes in beating cardiomyocytes in a standard culture dish. Various cardiomyocyte contraction parameters and oxygen consumption based on cell respiration could be determined from SECM data. We also confirmed that cellular changes induced by adding the cardiotonic agent digoxin were conveniently monitored by this SECM system. These results show that SECM can be a potentially powerful tool for use in drug development for cardiovascular diseases.     

"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.     

Macrophage adhesion on fibronectin evokes an increase in the elastic property of the cell membrane and cytoskeleton: an atomic force microscopy study

Interactions between cells and microenvironments are essential to cellular functions such as survival, exocytosis and differentiation. Cell adhesion to the extracellular matrix (ECM) evokes a variety of biophysical changes in cellular organization, including modification of the cytoskeleton and plasma membrane. In fact, the cytoskeleton and plasma membrane are structures that mediate adherent contacts with the ECM; therefore, they are closely correlated. Considering that the mechanical properties of the cell could be affected by cell adhesion-induced changes in the cytoskeleton, the purpose of this study was to investigate the influence of the ECM on the elastic properties of fixed macrophage cells using atomic force microscopy. The results showed that there was an increase (~50 %) in the Young’s modulus of macrophages adhered to an ECM-coated substrate as compared with an uncoated glass substrate. In addition, cytochalasin D-treated cells had a 1.8-fold reduction of the Young’s modulus of the cells, indicating the contribution of the actin cytoskeleton to the elastic properties of the cell. Our findings show that cell adhesion influences the mechanical properties of the plasma membrane, providing new information toward understanding the influence of the ECM on elastic alterations of macrophage cell membranes.     

小鼠胚胎心肌细胞的分离及电生理特性

本文旨在探索小鼠胚胎心肌细胞的分离方法并观察其电生理特性。应用胶原酶B消化法获得不同时期单个小鼠胚胎心肌细胞;利用全细胞膜片钳技术,记录胚胎心肌细胞的超极化激活的非选择性内向阳离子电流(If)和L-钙电流(ICa-L),并用电流钳记录其自发性动作电位。胚胎心肌细胞通过相差显微镜依据其形态和自发性收缩进行鉴定。本法分离所获得的胚胎心肌细胞容易进行全细胞膜片钳记录,可用于记录If,ICa-L．电流和自发性动作电位,己证实胚胎心肌细胞If和Ica-L的电生理特性与成年起搏细胞或心肌细胞相似。本实验建立的分离方法简单、稳定、有效、可靠,最早可获得8．5d的胚胎心肌细胞。胚胎心肌细胞的电生理记录为探索胚胎心肌细胞的电生理特性提供了一个可用的模型,并可能为某些心脏疾病产生的机制提供实验依据。     

Xenoreactive natural antibodies and induced antibodies — their effects on beating cardiomyocytes as a model of a xenograft

Xenotransplantation has been complicated by hyperacute rejection reactions, which are supposedly triggered by preformed natural antibodies (PNAb) of the recipient organism, whereas the role of antibodies specifically induced by previous antigen contact (IAb) is less clear.Primary cultures of spontaneously beating neonatal rat cardiomyocytes were used as a model of the heart to elaborate the effects of both PNAb and IAb from xenogeneic species and to investigate into their mechanisms of action. An experimental setup allowing for rapid medium exchange under continuous observation was employed.Sera containing PNAb reproducibly bring about a stereotype pattern of altered contractility including an initial increase in beating frequency followed by a temporary cessation of beating within the first minutes after administration. After recovery of spontaneous contractions, the cells within the monolayer exhibited a dissociation of the synchronicity of the beating persisting for several hours. The temporary pause in beating was prevented by a very high extracellular calcium concentration, but not by extracellular electrical stimulation sufficient to trigger contractions in control cells. Electrophysiological measurements carried out in adult ventricular guinea pig heart muscle cells under the same experimental conditions revealed an increase of the excitation threshold of the cells after application of sera containing PNAb due to an enhanced input resistance. These results indicate that the effect of PNAb is the consequence rather of a generally reduced excitability of the cell than of the inhibition of a singular ionic conductance. After specific absorption of PNAb directed against rat antigens beating of neonatal rat cardiomyocytes ensued without interruption. Sera specimens devoid of complement produced similar effects on contractility, although the duration of the standstill period was significantly shorter. The increase in input resistance visualized in guinea pig myocytes was absent after removing PNAb against guinea pig antigens but not after absorbing PNAb directed against rat epitopes.Signs of a permanent cytotoxicity after the administration of PNAb were lacking in all experiments.IAb against rat heart tissue raised in rabbits stopped the contractions of neonatal rat cardiomyocytes within 30 min after administration irreversibly and lead concentration-dependently to a destruction of the cells. (Mol Cell Biochem 160/161: 315–324, 1996)     

Cell culture of bivalves: tool for the study of the effects of environmental stressors.

Spontaneous beating cells can be isolated from the heart of the oyster (Crassostrea gigas) and cultured for more than two months. They form adherent contractile networks in culture conditions. They show muscarinic and beta-adrenergic reactivity thus showing that they are functional cardio-myocytes: Acetylcholine induced a dose dependent decrease in spontaneous beating rate via an increase in potassium conductance, this effect being blocked by atropine. Epinephrine induced a dramatic increase in calcium conductance which was blocked by high concentrations of propranolol but not by sotalol and reversed by verapamil. Tributyltin and cadmium induced a dose and time dependent decrease mainly in inward ionic conductances, leading to a decrease or even a total suppression of the beating rate. Present study indicates that this model could be used as a sensitive test to study the effects of some marine pollutants at the cellular level in molluscs.     

Experimental and Computational Investigation of the Role of Stress Fiber Contractility in the Resistance of Osteoblasts to Compression

The mechanical behavior of the actin cytoskeleton has previously been investigated using both experimental and computational techniques. However, these investigations have not elucidated the role the cytoskeleton plays in the compression resistance of cells. The present study combines experimental compression techniques with active modeling of the cell’s actin cytoskeleton. A modified atomic force microscope is used to perform whole cell compression of osteoblasts. Compression tests are also performed on cells following the inhibition of the cell actin cytoskeleton using cytochalasin-D. An active bio-chemo-mechanical model is employed to predict the active remodeling of the actin cytoskeleton. The model incorporates the myosin driven contractility of stress fibers via a muscle-like constitutive law. The passive mechanical properties, in parallel with active stress fiber contractility parameters, are determined for osteoblasts. Simulations reveal that the computational framework is capable of predicting changes in cell morphology and increased resistance to cell compression due to the contractility of the actin cytoskeleton. It is demonstrated that osteoblasts are highly contractile and that significant changes to the cell and nucleus geometries occur when stress fiber contractility is removed.     

原子力显微镜在细胞弹性研究中的应用

细胞表面的力学性质会随着细胞所处环境的不同而发生改变,它的变化间接反映出胞内复杂的生理过程。原子力显微镜（atomic force microscope,AFM）能以高的灵敏度和分辨率检测活体细胞,通过利用赫兹模型分析力曲线可以获得细胞的弹性信息。本文简介了原子力显微镜的工作原理与工作模式,着重介绍利用AFM力曲线检测细胞弹性的方法及其在细胞运动、细胞骨架、细胞黏附、细胞病理等方面的应用成果,表明AFM已经成为细胞弹性研究中十分重要的显微技术。     

Dynamic myocardial contractile parameters from left ventricular pressure-volume measurements

A new dynamic model of left ventricular (LV) pressure-volume relationships in beating heart was developed by mathematically linking chamber pressure-volume dynamics with cardiac muscle force-length dynamics. The dynamic LV model accounted for >80% of the measured variation in pressure caused by small-amplitude volume perturbation in an otherwise isovolumically beating, isolated rat heart. The dynamic LV model produced good fits to pressure responses to volume perturbations, but there existed some systematic features in the residual errors of the fits. The issue was whether these residual errors would be damaging to an application where the dynamic LV model was used with LV pressure and volume measurements to estimate myocardial contractile parameters. Good agreement among myocardial parameters responsible for response magnitude was found between those derived by geometric transformations of parameters of the dynamic LV model estimated in beating heart and those found by direct measurement in constantly activated, isolated muscle fibers. Good agreement was also found among myocardial kinetic parameters estimated in each of the two preparations. Thus the small systematic residual errors from fitting the LV model to the dynamic pressure-volume measurements do not interfere with use of the dynamic LV model to estimate contractile parameters of myocardium. Dynamic contractile behavior of cardiac muscle can now be obtained from a beating heart by judicious application of the dynamic LV model to information-rich pressure and volume signals. This provides for the first time a bridge between the dynamics of cardiac muscle function and the dynamics of heart function and allows a beating heart to be used in studies where the relevance of myofilament contractile behavior to cardiovascular system function may be investigated.     

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.     

A three-dimensional model of fluid-structural interactions for quantifying the contractile force for cardiomyocytes on hybrid biopolymer microcantilever

Quantitatively analysis of the contractility of cardiomyocytes is important for understanding the mechanism of heart failure as well as the molecular alterations in diseased heart cells. This paper presents a realistic computational model, which considers the three-dimensional fluid-structural interactions (FSI), to quantify the contractile force of cardiomyocytes on hybrid biopolymer microcantilevers. Prior to this study, only static modeling of the microscale cellular force has been reported. This study modeled the dynamics of cardiomyocytes on microcantilevers in a medium using the FSI. This realistic model was compared with static finite element modeling (FEM) analysis and the Stoney's equation-based analytical solution, and was validated by the deflections of the microcantilevers in the experimental results. Using harmonic response analysis in FSI modeling, the motion of a hybrid biopolymer microcantilever in the medium was identified as a second-order system and the influence of the dynamics of cardiomyocytes could be evaluated quantitatively.     

Isolation,Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca²⁺ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.     

Subcellular compartmentalization of myosin isoforms in embryonic chick heart ventricle myocytes during cytokinesis

Embryonic chick heart ventricle myocytes retain the ability to alternate between proliferation and functional differentiation. A cytoplasmic isoform of myosin is present in cleavage furrows of various nonmuscle cells during cytokinesis, whereas one or more of the cardiac myosin isoforms are localized in sarcomeres of beating cardiomyocytes. Antibodies were employed to reveal the subcellular localizations of cytoplasmic and cardiac myosin isoforms in embryonic chick ventricle cardiomyocytes during cytokinesis. Monoclonal anticytoplasmic myosin antibodies were prepared against myosin purified from brains of 1-day-posthatched chickens and shown to react with chick brain myosin heavy chain by Western blots and/or ELISA tests. One monoclonal antibrain myosin antibody also cross-reacted with chick cardiac myosin but not with skeletal or smooth muscle myosins. Two antichick cardiac myosin monoclonal antibodies and one antichick skeletal myosin polyclonal antibody that cross-reacts with cardiac myosin were employed to identify cardiac sarcomeric myosin. Cells were isolated from day 8 embryonic chick heart ventricles, enriched for myocytes, grown in vitro for 3 days, and then examined by immunofluorescence techniques. Monoclonal antibodies against cytoplasmic myosin preferentially localized in the cleavage furrows of both cardiofibroblasts and cardiomyocytes in all stages of cytokinesis. In contrast, antibodies that recognize cardiac myosin were distributed throughout cardiomyocytes during early stages of cytokinesis, but became progressively excluded from the furrow area during middle and late stages of cytokinesis. These data suggest that in cells that contain both cytoplasmic and sarcomeric myosin isoforms, only cytoplasmic myosin isoforms are mobilized to from the contractile ring for cytokinesis.     

Cocultures of fetal and adult cardiomyocytes yield rhythmically beating rod shaped heart cells from adult rats

Summary Different models of isolated cardiomyocytes are generally used for biochemical, biophysical, and pharmacological studies. Fetal cardiomyocytes can be easily cultured for several weeks regaining their ability for rhythmical and synchronous contractions. For investigations, differentiated myocytes derived from adult hearts are closer to the in situ situation. Unfortunately, these cells at best exhibit irregular and asynchronous contractions at very low frequencies. Already 1 d after seeding calcium-tolerant rod-shaped adult cardiomyocytes on a suitable substrate, the differentiated cells begin to dedifferentiate forming a confluent monolayer. After 7–10 d their beating activities are like those of fetal cells. Therefore, we tried to combine the advantages of both cell types to achieve fully differentiated cardiomyocytes, rod-shaped and rhythmically beating, isolated from adult hearts. Using contractile fetal cells as a substrate for the adult cardiomyocytes, freshly seeded differentiated adult myocytes are paced by the contraction frequency of the fetal monolayer. As a consequence, the rod-shaped adult cardiomyocytes reach frequencies of more than 140 cycles/min without external electrical stimulation. This model enables us to study cardiomyocytes in a state very similar to the in situ situation with respect to morphology, integrity, and contractile behavior. An abstract of this article was previously published in Eur. J. Cell Biol. 57 (Suppl.36): 86; 1992.     

3D-model of adult cardiac stem cells promotes cardiac differentiation and resistance to oxidative stress

The regenerative inadequacy of the injured myocardium leads to adverse remodeling, cardiac dysfunction, and heart disease. Stem cell-replacement of damaged myocardium faces major challenges such as inappropriate differentiation, cellular uncoupling, scar formation, and accelerated apoptosis of transplanted cells. These challenges can be met by engineering an in vitro system for delivering stem cells capable of cardiac differentiation, tissue integration, and resistance to oxidative stress. In this study, we describe the formation of three-dimensional (3D) cell aggregates ("cardiospheres") by putative stem cells isolated from adult dog myocardium using poly-L-ornithine. De novo formation of cardiospheres in growth factor-containing medium occurred over a period of 2-3 weeks, but accelerated to 2-3 days when seeded on poly-L-ornithine. Older cardiospheres developed foci of "beating" cells upon co-culture with rat neonatal cardiomyocytes. Cardiospheres contained cells that exhibited characteristics of undifferentiated cells; differentiating cardiomyocytes with organized contractile machinery; and vascular cells capable of forming "vessel-like" networks. They exhibited strong resistance to elevated concentrations of hydrogen peroxide in culture and survived subcutaneous injections without undergoing neoplastic transformation up to 3 weeks post-transplantation. These findings suggest that cardiospheres are potentially useful for delivering functional stem cells to the damaged heart.     

Current methods for the maturation of induced pluripotent stem cellderived cardiomyocytes

Induced pluripotent stem cells(iPSCs) were first generated by Yamanaka and colleagues over a decade ago. Since then, iPSCs have been successfully differentiated into many distinct cell types, enabling tissue-, disease-, and patientspecific in vitro modelling. Cardiovascular disease is the greatest cause of mortality worldwide but encompasses rarer disorders of conduction and myocardial function for which a cellular model of study is ideal. Although methods to differentiate iPSCs into beating cardiomyocytes(iPSC-CMs) have recently been adequately optimized and commercialized, the resulting cells remain largely immature with regards to their structure and function,demonstrating fetal gene expression, disorganized morphology, reliance on predominantly glycolytic metabolism and contractile characteristics that differ from those of adult cardiomyocytes. As such, disease modelling using iPSC-CMs may be inaccurate and of limited utility. However, this limitation is widely recognized, and numerous groups have made substantial progress in addressing this problem. This review highlights successful methods that have been developed for the maturation of human iPSC-CMs using small molecules,environmental manipulation and 3-dimensional(3 D) growth approaches.     

Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes

During early apoptosis, adult cardiomyocytes show unusual beating, suggesting possible participation of abnormal Ca(2+) transients in initiation of apoptotic processes in this cell type. Simultaneously with the beating, these cells show dynamic structural alteration resulting from cytoskeletal disintegration that is quite rapid. Because of the specialized structure and extensive cytoskeleton of cardiomyocytes, we hypothesized that its degradation in so short a time would require a particularly efficient mechanism. To better understand this mechanism, we used serial video microscopy to observe beta-adrenergic stimulation-induced apoptosis in isolated adult rat cardiomyocytes while simultaneously recording intracellular Ca(2+) concentration and cell length. Trains of Ca(2+) transients and corresponding rhythmic contractions and relaxations (beating) were observed in apoptotic cells. Frequencies of Ca(2+) transients and beating gradually increased with time and were accompanied by cellular shrinkage. As the cells shrank, amplitudes of Ca(2+) transients declined and diastolic intracellular Ca(2+) concentration increased until the transients were lost. Beating and progression of apoptosis were significantly inhibited by antagonists against the L-type Ca(2+) channel (nifedipine), ryanodine receptor (ryanodine), inositol 1,4,5-trisphosphate receptor (heparin), sarco(endo)plasmic Ca(2+)-ATPase (thapsigargin), and Na(+)/Ca(2+) exchanger (KB-R7943). Electron-microscopic examination of beating cardiomyocytes revealed progressive breakdown of Z disks. Immunohistochemical analysis and Western blot confirmed that disappearance of Z disk constituent proteins (alpha-actinin, desmin, and tropomyosin) preceded degradation of other cytoskeletal proteins. It thus appears that, in adult cardiomyocyte apoptosis, Ca(2+) transients mediate apoptotic beating and efficient sarcomere destruction initiated by Z disk breakdown.     

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.