Supplementing Soy-Based Diet with Creatine in Rats: Implications for Cardiac Cell Signaling and Response to Doxorubicin.

Nutritional habits can have a significant impact on cardiovascular health and disease. This may also apply to cardiotoxicity caused as a frequent side effect of chemotherapeutic drugs, such as doxorubicin (DXR). The aim of this work was to analyze if diet, in particular creatine (Cr) supplementation, can modulate cardiac biochemical (energy status, oxidative damage and antioxidant capacity, DNA integrity, cell signaling) and functional parameters at baseline and upon DXR treatment. Here, male Wistar rats were fed for 4 weeks with either standard rodent diet (NORMAL), soy-based diet (SOY), or Cr-supplemented soy-based diet (SOY + Cr). Hearts were either freeze-clamped in situ or following ex vivo Langendorff perfusion without or with 25 µM DXR and after recording cardiac function. The diets had distinct cardiac effects. Soy-based diet (SOY vs. NORMAL) did not alter cardiac performance but increased phosphorylation of acetyl-CoA carboxylase (ACC), indicating activation of rather pro-catabolic AMP-activated protein kinase (AMPK) signaling, consistent with increased ADP/ATP ratios and lower lipid peroxidation. Creatine addition to the soy-based diet (SOY + Cr vs. SOY) slightly increased left ventricular developed pressure (LVDP) and contractility dp/dt, as measured at baseline in perfused heart, and resulted in activation of the rather pro-anabolic protein kinases Akt and ERK. Challenging perfused heart with DXR, as analyzed across all nutritional regimens, deteriorated most cardiac functional parameters and also altered activation of the AMPK, ERK, and Akt signaling pathways. Despite partial reprogramming of cell signaling and metabolism in the rat heart, diet did not modify the functional response to supraclinical DXR concentrations in the used acute cardiotoxicity model. However, the long-term effect of these diets on cardiac sensitivity to chronic and clinically relevant DXR doses remains to be established.

Role of Cardiac AMP-Activated Protein Kinase in a Non-pathological Setting: Evidence From Cardiomyocyte-Specific, Inducible AMP-Activated Protein Kinase α1α2-Knockout Mice.

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis under conditions of energy stress. Though heart is one of the most energy requiring organs and depends on a perfect match of energy supply with high and fluctuating energy demand to maintain its contractile performance, the role of AMPK in this organ is still not entirely clear, in particular in a non-pathological setting. In this work, we characterized cardiomyocyte-specific, inducible AMPKα1 and α2 knockout mice (KO), where KO was induced at the age of 8 weeks, and assessed their phenotype under physiological conditions. In the heart of KO mice, both AMPKα isoforms were strongly reduced and thus deleted in a large part of cardiomyocytes already 2 weeks after tamoxifen administration, persisting during the entire study period. AMPK KO had no effect on heart function at baseline, but alterations were observed under increased workload induced by dobutamine stress, consistent with lower endurance exercise capacity observed in AMPK KO mice. AMPKα deletion also induced a decrease in basal metabolic rate (oxygen uptake, energy expenditure) together with a trend to lower locomotor activity of AMPK KO mice 12 months after tamoxifen administration. Loss of AMPK resulted in multiple alterations of cardiac mitochondria: reduced respiration with complex I substrates as measured in isolated mitochondria, reduced activity of complexes I and IV, and a shift in mitochondrial cristae morphology from lamellar to mixed lamellar-tubular. A strong tendency to diminished ATP and glycogen level was observed in older animals, 1 year after tamoxifen administration. Our study suggests important roles of cardiac AMPK at increased cardiac workload, potentially limiting exercise performance. This is at least partially due to impaired mitochondrial function and bioenergetics which degrades with age.

Activated Cardiac Fibroblasts Control Contraction of Human Fibrotic Cardiac Microtissues by a ß-Adrenoreceptor-Dependent Mechanism.

Cardiac fibrosis represents a serious clinical problem. Development of novel treatment strategies is currently restricted by the lack of the relevant experimental models in a human genetic context. In this study, we fabricated self-aggregating, scaffold-free, 3D cardiac microtissues using human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes and human cardiac fibroblasts. Fibrotic condition was obtained by treatment of cardiac microtissues with profibrotic cytokine transforming growth factor ß1 (TGF-ß1), preactivation of foetal cardiac fibroblasts with TGF-ß1, or by the use of cardiac fibroblasts obtained from heart failure patients. In our model, TGF-ß1 effectively induced profibrotic changes in cardiac fibroblasts and in cardiac microtissues. Fibrotic phenotype of cardiac microtissues was inhibited by treatment with TGF-ß-receptor type 1 inhibitor SD208 in a dose-dependent manner. We observed that fibrotic cardiac microtissues substantially increased the spontaneous beating rate by shortening the relaxation phase and showed a lower contraction amplitude. Instead, no changes in action potential profile were detected. Furthermore, we demonstrated that contraction of human cardiac microtissues could be modulated by direct electrical stimulation or treatment with the ß-adrenergic receptor agonist isoproterenol. However, in the absence of exogenous agonists, the ß-adrenoreceptor blocker nadolol decreased beating rate of fibrotic cardiac microtissues by prolonging relaxation time. Thus, our data suggest that in fibrosis, activated cardiac fibroblasts could promote cardiac contraction rate by a direct stimulation of ß-adrenoreceptor signalling. In conclusion, a model of fibrotic cardiac microtissues can be used as a high-throughput model for drug testing and to study cellular and molecular mechanisms of cardiac fibrosis.

3D Co-culture of hiPSC-Derived Cardiomyocytes With Cardiac Fibroblasts Improves Tissue-Like Features of Cardiac Spheroids.

Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions. Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture. Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

3D Cardiac Cell Culture: A Critical Review of Current Technologies and Applications.

Three-dimensional (3D) cell culture is often mentioned in the context of regenerative medicine, for example, for the replacement of ischemic myocardium with tissue-engineered muscle constructs. Additionally, 3D cell culture is used, although less commonly, in basic research, toxicology, and drug development. These applications have recently benefited from innovations in stem cell technologies allowing the mass-production of hiPSC-derived cardiomyocytes or other cardiovascular cells, and from new culturing methods including organ-on-chip and bioprinting technologies. On the analysis side, improved sensors, computer-assisted image analysis, and data collection techniques have lowered the bar for switching to 3D cell culture models. Nevertheless, 3D cell culture is not as widespread or standardized as traditional cell culture methods using monolayers of cells on flat surfaces. The many possibilities of 3D cell culture, but also its limitations, drawbacks and methodological pitfalls, are less well-known. This article reviews currently used cardiovascular 3D cell culture production methods and analysis techniques for the investigation of cardiotoxicity, in drug development and for disease modeling.

Measurement of Contractility and Calcium Release in Cardiac Spheroids.

There is a need for organotypic in vitro models that resemble the native tissue in functionality and tissue architecture for disease models and drug development. To this end, many 3D culture formats have been developed over time. Among the most often used type is the scaffold-free multicellular aggregate, also called spheroid, that forms by self-assembly. However, working with 3D cultures can be challenging because single cells are not as accessible as in 2D cultures and standard lab procedures must be adapted or replaced altogether. This chapter describes methods to create cardiac spheroids consisting of human iPSC-derived cardiomyocytes and cardiac fibroblasts and how to measure contractility or calcium signals using quantitative video analysis and confocal microscopy. Emphasis is on the particular challenges that 3D cultures pose and on affordable methods that do not require specialized equipment.

Characterization of cytoskeleton features and maturation status of cultured human iPSC-derived cardiomyocytes.

Recent innovations in stem cell technologies and the availability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have opened new possibilities for studies and drug testing on human cardiomyocytes in vitro. Still, there are concerns about the precise nature of such 'reprogrammed' cells. We have performed an investigation using immunocytochemistry and confocal microscopy on several cellular features using commercially available hiPSC-CMs. For some selected developmentally regulated or cardiac chamber-specific proteins, we have compared the results from hiPSC-derived cardiomyocytes with freshly isolated, ventricular cardiomyocytes from adult rats. The results show that all typical cardiac proteins are expressed in these hiPSC-CMs. Furthermore, intercalated disc-like structures, calcium cycling proteins, and myofibrils are present. However, some of these proteins are only known from early developmental stages of the ventricular myocardium or the diseased adult heart. A heterogeneous expression pattern in the cell population was noted for some muscle proteins, such as for myosin light chains, or incomplete organization in sarcomeres, such as for telethonin. These observations indicate that hiPSC-CMs can be considered genuine human cardiomyocytes of an early developmental state. The here described marker proteins of maturation may become instrumental in future studies attempting the improvement of cardiomyocyte in vitro models.

3D culture for cardiac cells.

This review discusses historical milestones, recent developments and challenges in the area of 3D culture models with cardiovascular cell types. Expectations in this area have been raised in recent years, but more relevant in vitro research, more accurate drug testing results, reliable disease models and insights leading to bioartificial organs are expected from the transition to 3D cell culture. However, the construction of organ-like cardiac 3D models currently remains a difficult challenge. The heart consists of highly differentiated cells in an intricate arrangement.Furthermore, electrical "wiring", a vascular system and multiple cell types act in concert to respond to the rapidly changing demands of the body. Although cardiovascular 3D culture models have been predominantly developed for regenerative medicine in the past, their use in drug screening and for disease models has become more popular recently. Many sophisticated 3D culture models are currently being developed in this dynamic area of life science. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Development and Characterization of a Scaffold-Free 3D Spheroid Model of Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes.

Cardiomyocytes (CMs) are terminally differentiated cells in the adult heart, and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSCs) of human origin was developed and characterized. Human CMs derived from iPSCs were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. Two-dimensional cultures were examined using immunofluorescence microscopy and western blotting, while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to prohypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived CMs formed spheroidal MTs within 4 days, showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca(2+) release, and extracellular calcium levels. A three-dimensional culture using iPSC-derived human CMs provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing a new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

Effects of doxorubicin cancer therapy on autophagy and the ubiquitin-proteasome system in long-term cultured adult rat cardiomyocytes.

The clinical use of anthracyclines in cancer therapy is limited by dose-dependent cardiotoxicity that involves cardiomyocyte injury and death. We have tested the hypothesis that anthracyclines affect protein degradation pathways in adult cardiomyocytes. To this aim, we assessed the effects of doxorubicin (Doxo) on apoptosis, autophagy and the proteasome/ubiquitin system in long-term cultured adult rat cardiomyocytes. Accumulation of poly-ubiquitinated proteins, increase of cathepsin-D-positive lysosomes and myofibrillar degradation were observed in Doxo-treated cardiomyocytes. Chymotrypsin-like activity of the proteasome was initially increased and then inhibited by Doxo over a time-course of 48 h. Proteasome 20S proteins were down-regulated by higher doses of Doxo. The expression of MURF-1, an ubiquitin-ligase specifically targeting myofibrillar proteins, was suppressed by Doxo at all concentrations measured. Microtubule-associated protein 1 light chain 3B (LC3)-positive punctae and both LC3-I and -II proteins were induced by Doxo in a dose-dependent manner, as confirmed by using lentiviral expression of green fluorescence protein bound to LC3 and live imaging. The lysosomotropic drug chloroquine led to autophagosome accumulation, which increased with concomitant Doxo treatment indicating enhanced autophagic flux. We conclude that Doxo causes a downregulation of the protein degradation machinery of cardiomyocytes with a resulting accumulation of poly-ubiquitinated proteins and autophagosomes. Although autophagy is initially stimulated as a compensatory response to cytotoxic stress, it is followed by apoptosis and necrosis at higher doses and longer exposure times. This mechanism might contribute to the late cardiotoxicity of anthracyclines by accelerated aging of the postmitotic adult cardiomyocytes and to the susceptibility of the aging heart to anthracycline cancer therapy.

Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes.

This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1α stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.

Cancer therapy-associated cardiotoxicity and signaling in the myocardium.

The cardiotoxic potential of cytotoxic cancer chemotherapy is well known. Prime examples are the anthracyclines, which are highly efficacious agents for hemopoietic malignancies and solid tumors, but their clinical use is limited primarily by cardiotoxicity. Besides the conventional chemotherapeutics, new cancer drugs were developed in the last decade with the goal to specifically inhibit selected molecular targets such as growth factor receptors or intracellular tyrosine kinases in cancer cells. However, the outcome of combining conventional and newer cancer therapies could have unexpected side effects not anticipated so far and the long-term outcome is not known. Sometimes, however, unexpected side effects also shed light on previously unknown physiological functions. For example, the anti-HER2 cancer therapeutic trastuzumab (Herceptin), which can induce cardiac dysfunction, has demonstrated the importance of the ErbB/neuregulin signaling system in the adult heart. Subsequently, the role of endothelial-myocardial communication in maintaining phenotype and survival of adult cardiomyocytes has increasingly been recognized.

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.

Inhibition of ErbB2 by receptor tyrosine kinase inhibitors causes myofibrillar structural damage without cell death in adult rat cardiomyocytes.

Inhibition of ErbB2 (HER2) with monoclonal antibodies, an effective therapy in some forms of breast cancer, is associated with cardiotoxicity, the pathophysiology of which is poorly understood. Recent data suggest, that dual inhibition of ErbB1 (EGFR) and ErbB2 signaling is more efficient in cancer therapy, however, cardiac safety of this therapeutic approach is unknown. We therefore tested an ErbB1-(CGP059326) and an ErbB1/ErbB2-(PKI166) tyrosine kinase inhibitor in an in-vitro system of adult rat ventricular cardiomyocytes and assessed their effects on 1. cell viability, 2. myofibrillar structure, 3. contractile function, and 4. MAPK- and Akt-signaling alone or in combination with Doxorubicin. Neither CGP nor PKI induced cardiomyocyte necrosis or apoptosis. PKI but not CGP caused myofibrillar structural damage that was additive to that induced by Doxorubicin at clinically relevant doses. These changes were associated with an inhibition of excitation-contraction coupling. PKI but not CGP decreased p-Erk1/2, suggesting a role for this MAP-kinase signaling pathway in the maintenance of myofibrils. These data indicate that the ErbB2 signaling pathway is critical for the maintenance of myofibrillar structure and function. Clinical studies using ErbB2-targeted inhibitors for the treatment of cancer should be designed to include careful monitoring for cardiac dysfunction.

Biosynthesis and expression of VE-cadherin is regulated by the PI3K/mTOR signaling pathway.

Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling.

Pathophysiology and diagnosis of cancer drug induced cardiomyopathy.

The clinical manifestations of anti-cancer drug associated cardiac side effects are diverse and can range from acutely induced cardiac arrhythmias to Q-T interval prolongation, changes in coronary vasomotion with consecutive myocardial ischemia, myocarditis, pericarditis, severe contractile dysfunction, and potentially fatal heart failure. The pathophysiology of these adverse effects is similarly heterogeneous and the identification of potential mechanisms is frequently difficult since the majority of cancer patients is not only treated with a multitude of cancer drugs but might also be exposed to potentially cardiotoxic radiation therapy. Some of the targets inhibited by new anti-cancer drugs also appear to be important for the maintenance of cellular homeostasis of normal tissue, in particular during exposure to cytotoxic chemotherapy. If acute chemotherapy-induced myocardial damage is only moderate, the process of myocardial remodeling can lead to progressive myocardial dysfunction over years and eventually induce myocardial dysfunction and heart failure. The tools for diagnosing anti-cancer drug associated cardiotoxicity and monitoring patients during chemotherapy include invasive and noninvasive techniques as well as laboratory investigations and are mostly only validated for anthracycline-induced cardiotoxicity and more recently for trastuzumab-associated cardiac dysfunction.

Inhibition of ErbB2/neuregulin signaling augments paclitaxel-induced cardiotoxicity in adult ventricular myocytes.

Paclitaxel (Taxol) has been successfully combined with the monoclonal antibody trastuzumab (Herceptin) in the treatment of ErbB2 overexpressing cancers. However, this combination therapy showed an unexpected synergistic increase in cardiac dysfunction. We have studied the mechanisms of paclitaxel/anti-ErbB2 cardiotoxicity in adult rat ventricular myocytes (ARVM). Myofibrillar organization was assessed by immunofluorescence microscopy and cell viability was tested by the TUNEL-, LDH- and MTT-assay. Oxidative stress was measured by DCF-fluorescence and myocyte contractile function by video edge-detection and fura-2 fluorescence. Treatment of ARVM with paclitaxel or antibodies to ErbB2 caused a significant increase in myofilament degradation, similarly as observed with an inhibitor of MAPK-signaling, but not apoptosis, necrosis or changes in mitochondrial activity. Paclitaxel-treatment and anti-ErbB2 reduced Erk1/2 phosphorylation. Paclitaxel increased diastolic calcium, shortened relaxation time and reduced fractional shortening in combination with anti-ErbB2. A minor increase in oxidative stress by paclitaxel or anti-ErbB2 was found. We conclude, that concomitant inhibition of ErbB2 receptors and paclitaxel treatment has an additive worsening effect on adult cardiomyocytes, mainly discernible in changes of myofibrillar structure and function, but in the absence of cell death. A potential mechanism is the modulation of the MAPK/Erk1/2 signaling by both drugs.

Neuregulin-1 beta attenuates doxorubicin-induced alterations of excitation-contraction coupling and reduces oxidative stress in adult rat cardiomyocytes.

Treatment of metastatic breast cancer with doxorubicin (Doxo) in combination with trastuzumab, an antibody targeting the ErbB2 receptor, results in an increased incidence of heart failure. Doxo therapy induces reactive oxygen species (ROS) and alterations of calcium homeostasis. Therefore, we hypothesized that neuregulin-1 beta (NRG), a ligand of the cardiac ErbB receptors, reduces Doxo-induced alterations of EC coupling by triggering antioxidant mechanisms. Adult rat ventricular cardiomyocytes (ARVM) were isolated and treated for 18-48 h. SERCA protein was analyzed by Western blot, EC coupling parameters by fura-2 and video edge detection, gene expression by RT-PCR, and ROS by DCF-fluorescence microscopy. At clinically relevant doses Doxo reduced cardiomyocytes contractility, SERCA protein and SR calcium content. NRG, similarly as the antioxidant N-acetylcystein (NAC), did not affect EC coupling alone, but protected against Doxo-induced damage. NRG and Doxo showed an opposite modulation of glutathione reductase gene expression. NRG, similarly as NAC, reduced peroxide- or Doxo-induced oxidative stress. Specific inhibitors showed, that the antioxidant action of NRG depended on signaling via the ErbB2 receptor and on the Akt- and not on the MAPK-pathway. Therefore, NRG attenuates Doxo-induced alterations of EC coupling and reduces oxidative stress in ARVM. Inhibition of the ErbB2/NRG signaling pathway by trastuzumab in patients concomitantly treated with Doxo might prevent beneficial effects of NRG in the myocardium.